Colocalization of glucagon-like peptide and glucagon immunoreactivities in pancreatic islets and intestine of salmonids

Summary Pancreatic islets of salmon contain at least two peptides of the glucagon family: 29-amino acid glucagon and 31-amino acid glucagon-like peptide (GLP). Both peptides were recently isolated from the pancreatic islets of coho salmon and sequenced (Plisetskaya et al. 1986). Antibodies generated against these two peptides and against human glucagon were used as immunocytochemical probes to investigate whether glucagon and GLP are processed in the same, or in different cell types in the pancreatic islets and the gut of salmon. Two salmonid species, rainbow trout and coho salmon, were studied. All islet A-cells in the two species were immunoreactive toward both anti-salmon (s)-glucagon and anti-s-GLP. Similar colocalization of glucagon and GLP immunoreactivities was found in open-type endocrine cells in mucosae of the small intestine (including the pyloric coecae) and the large intestine close to the vent of rainbow trout. None of the antibodies stained mucosal cells of the body of the stomach. These results suggest that in the pancreas and the gut of salmonid fish the same cells produce both glucagon and GLP. These peptides are most likely the products of a single gene coding for the preproglucagon sequence.     

Glucagon-like peptides activate hepatic gluconeogenesis

Piscine (anglerfish, catfish, coho salmon) glucagon-like peptides (GLPs), applied at 3.5 nM, stimulate (1.1-1.9-fold) flux through gluconeogenesis above control levels in isolated trout and salmon hepatocytes. Human GLP-1 and GLP-2 also activate gluconeogenesis, but to a lesser degree than their piscine counterparts. Minor increases of substrate oxidation are noticed at times of peak gluconeogenic activation through GLPs. These hormones, which are derived from the same precursor peptide as glucagon are more potent activators of gluconeogenesis than glucagon when applied at equimolar concentrations, and do not appear to employ cAMP or cGMP as the intracellular messenger in hepatic tissue.     

Characterization of coho salmon (Oncorhynchus kisutch) insulin

Insulin has been isolated from islet tissue of coho salmon (Oncorhynchus kisutch) by gel filtration and HPLC and the complete amino acid sequence has been determined. The sequence differs from bovine insulin at 14 sites but all interchanges are conservative from the viewpoint of preservation of conformation. A comparison of insulin sequences from other fish is presented. Salmon insulin cross-reacts very weakly with antiserum to bovine insulin and vice versa. A completely homologous radioimmunoassay has been developed and used to estimate the insulin in salmon islet tissue and in plasma. The hypoglycemic effect of salmon insulin in salmon was more pronounced and persisted longer than that caused by identical doses of bovine insulin.     

Charge inversion at position 68 of the glucagon and glucagon‐like peptide‐1 receptors supports selectivity in hormone action

Glucagon and glucagon‐like peptide‐1 (GLP‐1)are two structurally related hormones that acutely regulate glucose control in opposite directions through homologous receptors. The molecular basis for selectivity between these two hormones and their receptors is of physiological and medicinal importance. The application of co‐agonists to enhance body weight reduction and correct multiple abnormalities associated with the metabolic syndrome has recently been reported. Substitution of amino acids 16, 18, and 20 in glucagon with those found in GLP‐1 and exendin‐4 were identified as partial contributors to balanced, high potency receptor action. The amidation of the C‐terminus was an additional glucagon‐based structural change observed to be of seminal importance to discriminate recognition by both receptors. In this work, the molecular basis for receptor selectivity associated with differences in C‐terminal peptide sequence has been determined. A single charge inversion in glucagon and GLP‐1 receptor sequence at position 68* was determined to significantly alter hormone action. Changing E68* in GLP‐1R to the corresponding Lys of GCGR reduced receptor activity for natural GLP‐1 hormones by eightfold. The enhanced C‐terminal positive charges in GLP‐1 peptides favor the native receptor's negative charge at position 68*, while the unfavorable interaction with the C‐terminal acid of native glucagon is minimized by amidation. The extension of these observations to other glucagon‐related hormones such as oxyntomodulin and exendin, as well as other related receptors such as GIPR, should assist in the assembly of additional hormones with broadened pharmacology. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

Isolation and characterization of two coho salmon gonadotropins, GTH I and GTH II

Two gonadotropins, GTH I and GTH II, were isolated from pituitaries of spawning coho salmon (Oncorhynchus kisutch) using sequential extractions with ammonium acetate (pH 9.0) and 40% ethanol, precipitation with 80% ethanol, gel filtration chromatography (Sephadex G-100), anion-exchange chromatography (Mono-Q Sepharose), and gel filtration chromatography (Sephadex G-75). Coho salmon GTH I and GTH II stimulated steroidogenesis in vitro in a similar dose-dependent manner when incubated with either ovaries or testes of prepubertal coho salmon. An in vivo bioassay using coho salmon parr demonstrated that coho salmon GTH I and GTH II did not contain thyrotropic activity. Molecular weights were estimated by gel filtration chromatography to be 43,000 and 39,000 for GTH I and GTH II, respectively. Analysis of coho salmon GTH I and GTH II on reversed-phase high-performance liquid chromatography (rpHPLC) revealed that they consist of alpha and beta subunits with N-terminal amino acid residues of Tyr, Gly (alpha, beta of GTH I) and Tyr,Ser (alpha, beta of GTH II). Coho salmon GTH I-beta and GTH II-beta differed from each other in amino acid composition, N-terminal amino acids (Gly vs. Ser), and molecular weights in SDS-PAGE (19,000 vs. 20,000) and had a high degree of chemical similarity to chum salmon GTH I-beta and GTH II-beta, respectively. Specific rabbit antisera to the beta subunits of coho salmon GTH I and GTH II were generated. The observation of two GTHs with distinctly different chemical characteristics in coho salmon is similar to what has previously been found in chum salmon.     

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#.     

Chicken glucagon. Isolation and amino acid sequence studies.

Glucagon was isolated from a side fraction generated during the preparation of insulin and the new pancreatic peptide, avian pancreatic polypeptide from chicken pancreas. The immunological and biological properties are similar to those of beef-pork glucagon. The amino acid composition of chicken glucagon indicates that it contains 1 more serine residue than the porcine hormone and 1 less aspartic acid (asparagine) residue. Thus, chicken glucagon appears to be identical with turkey glucagon.     

Functional association of the N‐terminal residues with the central region in glucagon‐related peptides

GLP‐1 is an incretin peptide involved in the regulation of glucose metabolism and the glucose‐dependent stimulation of insulin secretion. Ex‐4 is a paralog of GLP‐1 that has comparable GLP‐1R potency but extended physiological action. GLP‐1 and Ex‐4 are helical peptides that share ～50% sequence homology but differ at several residues, notably the second amino acid which controls susceptibility to DPP‐IV cleavage. This single amino acid difference yields divergent receptor potency when studied in the context of the two hormone sequences. Ex‐4 uniquely tolerates Gly2 through select amino acid differences in the middle region of the peptide that are absent in GLP‐1. We report that substitution of Ex‐4 amino acids Glu16, Leu21, and Glu24 to the GLP‐1 sequence enabled Gly2 tolerance. The coordination of the N‐terminus with these central residues shows an interaction of substantial importance not only to DPP‐IV stability but also to receptor activation. Extension of this observation to glucagon‐based co‐agonist peptides showed different structural requirements for effective communication between the N‐terminus and the mid‐section of these peptides in achieving high potency agonism at the GLP‐1 and GCGRs. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Amino acid and hormonal control of macromolecular turnover in perfused rat liver. Evidence for selective autophagy

The control of RNA degradation by amino acids, insulin, and glucagon was investigated in perfused livers of fed rats previously labeled in vivo with [6-14C] orotic acid; rates were determined from the release of [14C]cytidine in the presence of 0.5 mM cytidine to suppress reutilization. Studies with cyclically perfused livers showed that plasma amino acids at 10 times (10X) normal concentrations inhibited RNA breakdown by 85%. Similar inhibition was obtained with a known regulatory amino acid mixture (Leu, Met, Pro, Trp, and His), whereas leucine alone (0.8 mM) decreased degradation by 47%. Perfusions carried out in the single-pass mode with graded levels of plasma amino acids revealed that the acceleration of RNA degradation over the full range of amino acid deprivation (0 to 10X normal levels) was the same as that for protein breakdown (3.19 and 3.15% h-1, respectively), and both were equally suppressed by insulin (2.4 micrograms h-1). Glucagon (10 micrograms h-1), though, was far less effective in stimulating RNA than protein turnover. A direct comparison of the two dose responses revealed a strong dissociation at 1 and 2 times normal amino acid levels. These findings support the notion that RNA and protein are degraded within a single macroautophagic compartment during amino acid and insulin deprivation. Glucagon, however, appeared to induce a second pathway in which the proportion of sequestered RNA to protein was selectively reduced. Electron micrographs showed that the ratio of vacuoles containing rough as compared with smooth endoplasmic reticulum was decreased by nearly 80% under these conditions.     

Preproglucagon gene expression in pancreas and intestine diversifies at the level of post-translational processing

Glucagon is a pancreatic hormone of 29 amino acids that regulates carbohydrate metabolism and glicentin is an intestinal peptide of 69 amino acids that contains the sequence of glucagon flanked by peptide extensions at the amino and carboxy termini. The glucagon gene encodes a precursor containing glucagon and two additional, structurally related, glucagon-like peptides separated by an intervening peptide. These peptides are encoded in separate exons. To determine whether the pancreatic and intestinal forms of glucagon arise by alternative RNA and/or protein processing, we used antisera to synthetic glucagon-like peptides and exon-specific, complementary oligonucleotides for analyses of proteins and mRNAs in pancreatic and intestinal extracts. Preproglucagon mRNAs are identical, but different and highly specific peptides are liberated in the two tissues. Immunocytochemistry shows colocalization of glucagon and the two glucagon-like peptides in identical cells. We conclude that diversification of preproglucagon gene expression occurs at the level of cell-specific post-translational processing.     

Molecular cloning and sequencing of coho salmon growth hormone cDNA

A cDNA library was constructed using mRNA isolated from coho salmon pituitaries. By employing rainbow trout growth hormone cDNA as a probe, the coho salmon cDNA was isolated and the complete nucleotide (nt) sequence determined. The coding region contains 630 nt while the 5'- and 3'-untranslated regions are 64 and 489 nt in length, respectively. Comparison of the noncoding regions of coho and chum salmon cDNAs reveal identity at the 5' end but significant variation in the 3' end. Chum salmon and rainbow trout have identical amino acid (aa) sequences, but coho salmon growth hormone has a sequence that differs by 6 of the 188 predicted aa. Since salmonids are tetraploid, this difference may be the result of either divergence of the same growth hormone locus or of variation between different loci. Comparisons of the cDNA restriction maps of these three fish species suggest the former possibility.     

Studies on Nα-acylated proteins: The N-terminal sequences of two muscle enolases

A standard procedure for the identification of the N-terminal amino acid in N alpha-acylated proteins has been developed. After exhaustive proteolysis, the amino acids with blocked alpha-amino groups are separated from positively charged, free amino acids by ion exchange chromatography and subjected to digestion with acylase I. Amino acid analysis before and after the acylase treatment identifies the blocked N-terminal amino acid. A survey of acylamino acid substrates showed that acylase will liberate all the common amino acids except Asp, Cys or Pro from their N-acetyl-and N-butyryl derivatives, and will also catalyze the hydrolysis of N-formyl-Met and N-myristyl-Val. Thus, the procedure cannot identify acylated Asp, Cys or Pro, nor, because of the ion exchange step, N alpha-acyl-derivatives of Arg, Lys or His. Whenever the protease treatment releases free acylamino acids, the remaining amino acids should be detected. When applied to several proteins, the procedure confirmed known N-terminal acylamino acids and identified acyl-Ser in enolases from chum and coho salmon muscle and in pyruvate kinase from rabbit muscle, and acyl-Thr in phosphofructokinase from rabbit muscle. The protease-acylase assay has been used to identify blocked peptides from CNBr- or protease-treated proteins. When such peptides were treated with 1 N HCl at 110 degrees for 10 min, sufficient yields of deacylated, mostly intact, peptide were obtained to permit direct automatic sequencing. The N-terminal sequences of rabbit muscle and coho salmon enolase were determined in this way and are compared to each other and to the sequence of yeast enolase.     

DPP‐IV‐resistant,long‐acting oxyntomodulin derivatives

Obesity is one of the major risk factors for type 2 diabetes, and the development of agents, that can simultaneously achieve glucose control and weight loss, is being actively pursued. Therapies based on peptide mimetics of the gut hormone glucagon‐like peptide 1 (GLP‐1) are rapidly gaining favor, due to their ability to increase insulin secretion in a strictly glucose‐dependent manner, with little or no risk of hypoglycemia, and to their additional benefit of causing a modest, but durable weight loss. Oxyntomodulin (OXM), a 37‐amino acid peptide hormone of the glucagon (GCG) family with dual agonistic activity on both the GLP‐1 (GLP1R) and the GCG (GCGR) receptors, has been shown to reduce food intake and body weight in humans, with a lower incidence of treatment‐associated nausea than GLP‐1 mimetics. As for other peptide hormones, its clinical application is limited by the short circulatory half‐life, a major component of which is cleavage by the enzyme dipeptidyl peptidase IV (DPP‐IV). SAR studies on OXM, described herein, led to the identification of molecules resistant to DPP‐IV degradation, with increased potency as compared to the natural hormone. Analogs derivatized with a cholesterol moiety display increased duration of action in vivo. Moreover, we identified a single substitution which can change the OXM pharmacological profile from a dual GLP1R/GCGR agonist to a selective GLP1R agonist. The latter finding enabled studies, described in detail in a separate study (Pocai A, Carrington PE, Adams JR, Wright M, Eiermann G, Zhu L, Du X, Petrov A, Lassman ME, Jiang G, Liu F, Miller C, Tota LM, Zhou G, Zhang X, Sountis MM, Santoprete A, Capitò E, Chicchi GG, Thornberry N, Bianchi E, Pessi A, Marsh DJ, SinhaRoy R. Glucagon‐like peptide 1/glucagon receptor dual agonism reverses obesity in mice. Diabetes 2009; 58: 2258–2266), which highlight the potential of GLP1R/GCGR dual agonists as a potentially superior class of therapeutics over the pure GLP1R agonists currently in clinical use. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

~(21)Ala定点突变胰高血糖素及结构与功能变化

 胰高血糖素是由 2 9个氨基酸组成的多肽激素 ,具有促糖元分解的生理功能 ,其拮抗剂有治疗糖尿病病人的潜在应用价值 .在获得重组胰高血糖素基因工程菌基础上 ,利用定点突变技术改造其第 2 1位氨基酸天冬氨酸为丙氨酸 ,并经DNA测序证明胰高血糖素基因发生了点突变 .用IPTG诱导表达后 ,经亲和层析和反相高效液相层析 ,纯化到突变型重组2 1Ala 胰高血糖素 .质谱测定分子量与理论值相符 .利用园二色谱比较重组胰高血糖素和突变的2 1Ala 胰高血糖素在TFE中的二级结构 ,发现胰高血糖素以α螺旋为主要二级结构 ,2 1Ala 胰高血糖素仍有α螺旋结构特征 ,并且含量有所增大 .利用兔升血糖试验 ,发现2 1Ala 胰高血糖素生物活性比重组胰高血糖素减少 51 % (P <0 .0 1 ) .显示天然胰高血糖素第 2 1位氨基酸天冬氨酸与形成α螺旋结构关系不大 ,但在发挥胰高血糖素的生物功能中有重要作用 ,与其可作为钙离子结合位点 ,参与胰高血糖素和受体结合的潜在功能密切相关 .     

Amino acid cues emanating from Pacific salmon eggs and ovarian fluid

The eggs of salmonid fishes are an important food source for many aquatic predators that detect eggs using olfaction. Moreover, chemicals from eggs and ovarian fluid aid sperm cells in detecting and locating eggs for fertilization, and ovarian fluid is attractive to conspecific males. Thus chemicals from eggs and ovarian fluid may facilitate reproduction but may also attract egg predators. The authors sampled mature females of three Pacific salmon species – Chinook (Oncorhynchus tshawytscha), coho (Oncorhynchus kisutch) and sockeye (Oncorhynchus nerka) – and determined the proportional representation of amino acids, potent fish odorants, from their eggs and ovarian fluid (Chinook and coho salmon only). They then tested juvenile coho salmon, an egg predator, for responses to ovarian fluid and egg odours using the electro-olfactogram (EOG) recording technique. The amino acid compositions of the salmon species were significantly and positively correlated with each other, and the interspecific differences were comparable to those between individuals of the same species. The egg water samples were, on average, dominated by lysine, alanine and glutamine (12.6%, 12.4% and 10.9%, respectively). The ovarian fluid samples were dominated by lysine (20.5%), followed by threonine (9.7%), glycine (9.2%) and arginine (8.8%). EOG recordings demonstrated the ability of juvenile coho salmon to detect the chemical traces of eggs and ovarian fluid. It is concluded that salmon eggs are a potent source of odours for potential predators but likely not highly differentiated among salmon species.     

Isolation and structures of glucagon and glucagon-like peptide from catfish pancreas

Both glucagon and the structurally similar glucagon-like peptide proteolytically derived from preproglucagon were purified from the endocrine pancreas of the channel catfish (Ictalurus punctata). This study represents the first report of the isolation of glucagon-like peptide from any source. Peptide sequences of glucagon-like peptide from other species have only been deduced from the cDNA sequences for preproglucagon. The sequence of the 34-residue glucagon-like peptide was found to be HADGTYTSDVSSYLQDQAAKDFITWLKSGQPKPE. Catfish glucagon-like peptide shares sequence identity at 26 of 31 residues with the putative glucagon-like peptide from anglerfish preproglucagon II. The mass of catfish glucagon-like peptide was found by fast atom bombardment-mass spectrometry to be 3785, identical with the value predicted by sequence analysis. This suggests that no post-translational modification occurs beyond proteolytic processing. The sequence of catfish glucagon was determined to be HSEGTFSNDYSKYLETRRAQDFVQWLM(N,S). Catfish glucagon exhibits a high degree of immunologic similarity with porcine glucagon by radioimmunoassay, whereas catfish glucagon-like peptide does not.     

Nucleotide sequence and growth hormone-regulated expression of salmon insulin-like growth factor I mRNA

Protein and cDNA sequence analysis have revealed that the insulin-like growth factor (IGF-I) has been highly conserved among several mammalian species. Using the combined techniques of polymerase chain reaction and molecular cloning, we have now obtained the cDNA sequence encoding preproIGF-I from a teleost species, Oncorhynchus kisutch (coho salmon). The 2020 nucleotide (nt) cloned cDNA sequence contains a 528 nt open reading frame encoding 176 amino acids in preproIGF-I and 175 nt and 1317 nt of flanking 5'- and 3'-untranslated regions, respectively. The deduced amino acid sequence of salmon IGF-I is highly conserved relative to its mammalian homologues and there are only 14 amino acid differences out of 70 between salmon and human IGF-I. Interestingly, the C-terminal E domain of salmon proIGF-I, which is presumed to be proteolytically cleaved during biosynthesis, also shows striking amino acid sequence homology with its mammalian counterpart, except for an internal 27 residue segment that is unique to salmon proIGF-I. Northern analysis revealed that salmon preproIGF-I mRNA consists predominantly of a single 3900 nt sized band although minor bands were also observed after prolonged autoradiographic exposure. The RNA analysis also revealed that the level of preproIGF-I mRNA is increased 6-fold in liver RNA isolated from salmon injected with bovine GH, as compared to untreated controls. These results demonstrate that the primary structure and regulated expression of IGF-I by GH have been conserved in teleosts.     

Effect of orally administered phenylalanine with and without glucose on insulin, glucagon and glucose concentrations.

OBJECTIVE: As part of our studies of the metabolic effects of ingested proteins, we are currently investigating the effects of ingestion of individual amino acids. The objective of the present study was to determine whether ingested phenylalanine stimulates insulin and/or glucagon secretion, and if phenylalanine ingested with glucose modifies the insulin, glucagon or glucose response to the ingested glucose. DESIGN: Six healthy subjects were tested on 4 separate occasions. Plasma phenylalanine, glucose, insulin, glucagon, and total alpha amino nitrogen (AAN) (i.e., total amino acids) concentrations were measured at various times during a 2.5 h period after ingestion of 1 mmol phenylalanine/kg lean body mass, 25 g glucose, 1 mmol phenylalanine/kg lean body mass+25 g glucose, or water only, given in random order. RESULTS: Following phenylalanine ingestion, the circulating phenylalanine concentration increased approximately 14 fold and remained elevated for the duration of the experiment. Glucagon and AAN increased, insulin increased modestly, and glucose was unchanged when compared to water ingestion. When glucose was ingested with phenylalanine, the circulating phenylalanine, glucagon, AAN, and insulin area responses were approximately the sum of the responses to phenylalanine alone and glucose alone. However, the plasma glucose area response was decreased 66% when phenylalanine was co-ingested with glucose. CONCLUSION: In summary, phenylalanine in an amount moderately greater than that in a large protein meal stimulates an increase in insulin and glucagon concentration. It markedly attenuates the glucose-induced rise in plasma glucose when ingested with glucose.     

Structural requirements of Bacillus subtilis alpha-amylase signal peptide for efficient processing: in vivo pulse-chase experiments with mutant signal peptides.

The Bacillus subtilis alpha-amylase signal peptide consists of 33 amino acids from its translation initiation site. To analyze the structural requirements for efficient processing of the signal peptide, single and repeated Ala-X-Ala sequences and their modifications were introduced into B. subtilis alpha-amylase signal peptides of different lengths and the mature thermostable alpha-amylase. Then the cleavage positions and processing rates of the signal peptides were analyzed by the NH2-terminal amino acid sequences of the exported thermostable alpha-amylases and by in vivo pulse-chase experiments. In B. subtilis, the most efficient cleavage site was located at the peptide bond between Ala-33 and amino acid X at position 34, even though Val-X-Ala and six repeating Ala-X-Ala sequences were present around the cleavage site. However, the cleavage site was shifted to the peptide bond between Ala-31 and amino acid X when Ala-33 was deleted, and it was also shifted to Ala-35 and X when Ala-33 was replaced with Val-33. The shorter signal peptide consisting of 31 amino acids reduced the processing rate and alpha-amylase production. In contrast, those signal peptides were cleaved preferentially at the peptide bond between Ala-31 and amino acid X in Escherichia coli. In addition to the presence of an Ala residue at the -1 amino acid position, the length of the signal peptide was another important requirement for efficient processing.