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.     

Singular contributions of fish neuroendocrinology to mammalian regulatory peptide research

During the past 20 years, several bioactive peptides have been identified in teleost fishes that subsequently have been shown to play important regulatory roles in mammalian physiology. The urophysis, corpuscles of Stannius and Brockmann body are anatomical structures particular to fish that have no obvious counterpart in mammals. Extracts and/or cDNA libraries prepared from these tissues have been used to identify for the first time urotensin II (U-II), urotensin-I (U-I), stanniocalcin and glucagon-like peptide-1 (GLP-1). Although U-II and U-I were originally regarded as exclusively the products of the teleost urophysis, the peptides have a wide phylogenetic distribution across the vertebrate lineage, including mammals. U-II is localized to motor neurones in the human spinal cord and is a potent vasoconstrictor that may be implicated in the pathogenesis of heart failure. The human ortholog of urotensin-I is urocortin which is synthesized in selected regions of the brain and is the endogenous ligand for the CRF type 2 receptor. Urocortin is believed to important in mediating the effects of stress on appetite. Stanniocalcin is involved in maintaining calcium and phosphate homeostasis in teleost fish. An ortholog of stanniocalcin has a widespread distribution in mammalian tissues and is postulated to regulate renal phosphate excretion and to protect neurons against damage during cerebral ischemia. The biological actions and therapeutic potential of GLP-1 in humans are now fully appreciated but the peptide was first identified as a domain in a preproglucagon cDNA prepared from anglerfish Brockmann bodies. In contrast to mammalian preproglucagons, GLP-1 is present in anglerfish preproglucagon as the bioactive, truncated sequence [corresponding to human GLP-1(7-37)] rather than the inactive, N-terminally extended form [corresponding to GLP-1(1-37)]. Failure to appreciate the significance of this fact retarded progress in the field for several years.     

The discovery of glucagon-like peptide 1

The discovery of glucagon-like peptide 1 (GLP-1) began more than two decades ago with the observations that anglerfish islet proglucagon messenger RNAs (mRNAs) contained coding sequences for two glucagon-related peptides arranged in tandem. Subsequent analyses revealed that mammalian proglucagon mRNAs encoded a precursor containing the sequence of pancreatic glucagon, intestinal glicentin and two glucagon-related peptides termed GLP-1 and GLP-2. Multidisciplinary approaches were then required to define the structure of biologically active GLP-1 7-36 amide and its role as an incretin, satiety hormone and, most recently, a neuroprotective peptide. This historial perspective outlines the use of traditional recombinant DNA approaches to derive the GLP-1 sequence and highlights the challenges and combination of clinical and basic science approaches required to define the physiology and pathophysiology of bioactive peptides discovered through genomics.     

GIP and GLP-1 as incretin hormones: lessons from single and double incretin receptor knockout mice

Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are gut-derived incretins secreted in response to nutrient ingestion. Both incretins potentiate glucose-dependent insulin secretion and enhance beta-cell mass through regulation of beta-cell proliferation, neogenesis and apoptosis. In contrast, GLP-1, but not GIP, inhibits gastric emptying, glucagon secretion, and food intake. Furthermore, human subjects with Type 2 diabetes exhibit relative resistance to the actions of GIP, but not GLP-1R agonists. The physiological importance of both incretins has been investigated through generation and analysis of incretin receptor knockout mice. Elimination of incretin receptor action in GIPR-/- or GLP-1R-/- mice produces only modest impairment in glucose homeostasis. Similarly, double incretin receptor knockout (DIRKO) mice exhibit normal body weight and normal levels of plasma glucagon and hypoglycemic responses to exogenous insulin. However, glucose-stimulated insulin secretion is significantly decreased following oral but not intraperitoneal glucose challenge in DIRKO mice and the glucose lowering actions of dipeptidyl peptidase-IV (DPP-IV) inhibitors are extinguished in DIRKO mice. Hence, incretin receptor signaling exerts physiologically relevant actions critical for glucose homeostasis, and represents a pharmacologically attractive target for development of agents for the treatment of Type 2 diabetes.     

Human glucagon-like peptides 1 and 2 activate rat brain adenylate cyclase

Two human glucagon-like peptides, GLP-1 and GLP-2, which are coencoded with pancreatic glucagon in the preproglucagon gene, do not significantly inhibit [125I]monoiodoglucagon binding to rat liver and brain membranes and do not activate adenylate cyclase in liver plasma membranes. Nevertheless, GLP-1 and GLP-2 were each found to be potent stimulators of both rat hypothalamic and pituitary adenylate cyclase. Only 30-50 pM concentrations of each peptide elicited half-maximal adenylate cyclase stimulation. Our data suggest that GLP-1 and GLP-2 may be neurotransmitters and/or neuroendocrine effectors, which would account for their high degree of sequence conservation through vertebrate evolution.     

Effects of substitution of N-terminal amino acid of glucagon-like peptide-1 (7-36) amide on food intake of the neonatal chick

Glucagon-like peptide-1 (GLP-1), a member of glucagon superfamily, is synthesized from a large precursor, preproglucagon, and has been postulated to be a novel incretin. Recently, it was reported that central administration of GLP-1 (7-36) amide decreased food intake in rats and chickens. Generally, the amino acid sequences of the glucagon superfamily members except for gastric inhibitory peptide and growth hormone-releasing factor are identical at N-terminal histidine. It is well known that the GLP-1 receptor is highly specific for GLP-1 and does not bind other peptides of the glucagon superfamily. The aim of this study was to elucidate whether central injection of substituted GLP-1 in which N-terminal histidine of mammalian GLP-1 (7-36) amide was replaced with tyrosine, inhibits food intake in the chick. Intracerebroventricular administration of substituted GLP-1 inhibits food intake in the chick, although the effect of substituted GLP-1 was 11 to 13 fold less than that of mammalian GLP-1 (7-36) amide. These results indicate that N-terminal histidine of GLP-1 (7-36) amide is important for efficacy, but not essential for its bioactivity.     

Isolation and structure of the principal products of preproglucagon processing, including an amidated glucagon-like peptide

The principal products derived from in vivo processing of anglerfish preproglucagon II were isolated and their structures determined. The structures were confirmed by a combination of automated Edman degradation, amino acid analysis, and fast atom bombardment mass spectrometry. The peptide corresponding to anglerfish preproglucagon II-(22-49) (numbering from the amino terminus of preproglucagon) was isolated intact and defines the site of signal cleavage to be between Gln-21 and Met-22. Glucagon from the anglerfish preproglucagon gene II was found to correspond to preproglucagon II-(52-80) (numbering from the amino terminus). Three forms of a glucagon-like peptide derived from preproglucagon II were also isolated. The structure of the longest form was consistent with the sequence of preproglucagon II-(89-122) deduced from the cDNA, His-Ala-Asp-Gly-Thr-Tyr-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Gln-Asp-Gln-Ala- Ala-Lys-Asp-Phe-Val-Ser-Trp-Leu-Lys-Ala-Gly-Arg-Gly-Arg-Arg-Glu. The carboxyl-terminal portion deduced from the cDNA remains intact in this form. A second form, preproglucagon II-(89-119) appears to result from proteolytic processing of the major form at the two adjacent arginine residues occurring at the carboxyl terminus. This second form has a glycine residue at its carboxyl terminus and is processed to the third form (preproglucagon II-(89-118)) which contains a carboxyl-terminal arginineamide. Radiolabeling studies in primary tissue culture support the observation that glucagon (preproglucagon II-(52-80], preproglucagon II-(89-122), and preproglucagon II-(89-119) are products of proglucagon processing in vivo.     

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.     

Incretin hormones in the treatment of type 2 diabetes. Part I: influence of insulinotropic gut-derived hormones (incretins) on glucose metabolism

Insulinotropic gut-derived hormones (incretins) play a significant role in the regulation of glucose homeostasis in healthy subjects and are responsible for 50-70% of insulin response to a meal. The main mediators of the incretin effect are glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1). However, in patients with type 2 diabetes the effect of incretins action is to a large extent impaired, which seems to explain disturbed secretional activity of beta cells in pancreatic islets. Detailed analysis of incretin defect proved that GIP secretion remains within physiological limits, whereas GLP-1 secretion is significantly decreased. Nevertheless, GLP-1 insulinotropic effect is preserved and GIP effect is significantly impaired. In consequence, substitutional GLP-1 administration aiming at the reduction of its deficiency, seems to be logical therapeutic management, because despite a physiologically retained quantity response from GIP, resistance to this peptide is frequently found. Therefore, particularly promising are the results of clinical studies with the use of GLP-1 analogues , GLP-1 receptors activation, as well as the inhibitors of dipeptidyl peptidase-IV (DPP IV), the enzyme responsible for incretin proteolysis, which restores the proper function of the intestinal-pancreatic axis in subjects with type 2 diabetes and creates new possibilities of a glycaemia reducing therapy and improvement in quality of life in this group of patients.     

Characterization of insulin and atypically processed proglucagon-derived peptides from the surinam toad Pipa pipa (Anura:Pipidae)

Electrospray mass spectrometry was used to identify insulin, glucagon and two peptides related to glucagon-like peptide-1 (GLP-1) in an extract of the pancreas of the Surinam toad, Pipa pipa, a species belonging to the same family as the African clawed frog, Xenopus laevis. Purification and characterization of the peptides established the primary structure of Pipa insulin as A-chain: GIVEQCCHSS(10)CTLLQLETYC(20) N and B-Chain: FSNQR LCGSH(10) LVEALHLVCG(20) DRGFFYYPKA(30). This amino acid sequence contains several substitutions (B5 His --> Arg, B16 Tyr --> His, A12 Ser --> Thr, A14 Tyr--> Leu, A18Asn --> Thr) of residues that have otherwise been quite strongly conserved during vertebrate evolution. Pipa glucagon comprises 37 amino acid residues (HSQGTFTSDY(10) SKYLDSRRAQ(20) DFVQWLMNTK(30)QSGGLSS) and the 29 amino-acid-residue peptide was not identified in the extract. In Xenopus and mammalian preproglucagons, the glucagon-29 sequence is followed by Lys-Arg which functions as a recognition site for a prohormone convertase. We propose that a point mutation in the gene encoding Pipa preproglucagon has transformed the Lys(30)-Arg(31) processing site into Lys-Gln with the result that the site in no longer recognized by the processing enzyme. In contrast, Pipa GLP-32 and GLP-37 are of the same molecular size as the corresponding peptides from Xenopus.     

Localization of glucagon-like peptide (GLP) immunoreactants in human gut and pancreas using light and electron microscopic immunocytochemistry

The distribution of peptide immunoreactivities predicted from the sequence of the human preproglucagon gene in enteroglucagon (EG; glicentin-like immunoreactant-containing) cells of the human gut and A cells of the pancreas has been determined by light and electron microscopic immunocytochemistry. At light microscopy the application of peroxidase-antiperoxidase and immunogold-silver staining methods has revealed that glucagon-like peptide (GLP-1 and GLP-2) immunoreactivities coexist with a glicentin-related immunodeterminant in human colorectal EG cells and pancreatic A cells. Using single and double colloidal gold probe electron immunocytochemistry, we have been able to show the coexistence of glicentin, GLP-1, and GLP-2 immunoreactivities within single EG cell secretory granules. No morphologic segregation of the proglucagon immunoreactants was observed in EG cells of the colonic mucosa. In pancreatic A cells we have localized GLP-1, GLP-2, and glucagon-[16-29] immunoreactivities solely to the electron-dense core of the secretory granules, whereas glicentin-related immunoreactivity was restricted to the electron-lucent halo. The results obtained in the present study have shown that the peptide immunoreactivities predicted from cDNA sequencing of the human preproglucagon gene are indeed expressed in colorectal EG and pancreatic A cells. The topographical segregation of immunoreactivities in the A cell secretory granule shows that antigenic determinants derived from the C-terminal portion of proglucagon are stored with glucagon in the core of the secretory granule.     

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 peptide-1 and the entero-insular axis in obese hyperglycaemic (ob/ob) mice

The intestines of obese hyperglycaemic (ob/ob) mice contain greatly increased amounts of glucagon-like immunoreactive peptides. To investigate their role in the increased activity of the entero-insular axis of these mice, the insulin-releasing effect of glucagon-like peptide-1 (GLP-1) was examined in 24 hour fasted 12-15 weeks old ob/ob mice under conditions of basal and elevated glycaemia. Compared with glucagon (100 micrograms/kg ip), which produce an approximately 3-fold increase in basal plasma glucose and insulin concentrations, GLP-1 (100 micrograms/kg ip) produce a very small (less than 1 fold) increase in plasma insulin, with no significant change in plasma glucose. The insulin-releasing effect of glucagon, but not GLP-1 was increased by administration in combination with glucose (2 g/kg ip). The results indicate that GLP-1, which exhibits considerable sequence homology with glucagon, exerts only a weak insulin-releasing effect without a significant hyperglycaemic effect in ob/ob mice. Thus GLP-1 is unlikely to be an important endocrine component of the two over-active entero-insular axis in ob/ob mice.     

Evolution of glucagon genes

Statistical analyses of DNA sequences of the preproglucagon genes from bovine, human, hamster, and anglerfish suggest that a gene duplication creating two anglerfish genes (AF I and II) occurred about 160 Myr ago, long after the separation of fish and mammals. The analyses further suggest that the internal duplication producing the glucagon and glucagon-like peptide II (GLP-II) regions occurred about 1.2 billion years ago, which would indicate that the GLP-II region was present in the ancestral anglerfish sequence but was silenced or deleted before the gene duplication separating AF I and II. The glucagon-like peptide I (GLP-I) was derived from a duplication of the ancestral glucagon region about 800 Myr ago. The rate of synonymous substitution in these genes is approximately 4.3 x 10(-9) substitutions per year per synonymous site. The rate of nonsynonymous substitution in the signal peptide region is about 1.1 x 10(-9) substitutions per year per nonsynonymous site, a high rate comparable to that in the C-peptide region of preproinsulin. The rate of nonsynonymous substitution in the glicentin-related pancreatic polypeptide (GRPP) region is 0.63 x 10(-9) for the comparisons between mammalian species and 1.8 x 10(-9) for the comparisons between fish and mammals; the moderate rate in mammals suggests a physiological role for GRPP. The glucagon region is extremely conservative; no nonsynonymous substitution is observed in the mammalian genes, and a nonsynonymous rate of 0.18 x 10(-9) was obtained from the comparisons between fish and mammals. In the GLP-I region, the rate of nonsynonymous substitution was estimated to be 0.08 x 10(-9) for the comparisons between mammalian species and 0.30 x 10(- 9) for the comparisons between fish and mammals. In the GLP-II region, the rate was estimated to be 0.25 x 10(-9) for the comparisons between mammalian species. Thus, GLP-I and II are also very conservative, which suggests an important physiological role for these peptides.      

Evolution of new hormone function: loss and gain of a receptor

The vertebrate proglucagon gene encodes three glucagon-like sequences (glucagon, glucagon-like peptide-1 [GLP-1], and glucagon-like peptide 2 [GLP-2]) that have distinct functions in regulating metabolism in mammals. In contrast, glucagon and GLP-1 have similar physiological actions in fish, that of mammalian glucagon. We have identified sequences similar to receptors for proglucagon-derived peptides from the genomes of two fish (pufferfish and zebrafish), a frog (Xenopus tropicalis), and a bird (chicken). Phylogenetic analysis of the receptor sequences suggested an explanation for the divergent function of GLP-1 in fish and mammals. The phylogeny of our predicted and characterized receptors for proglucagon-derived peptides demonstrate that receptors for glucagon, GLP-1, and GLP-2 have an origin before the divergence of fish and mammals; however, fish have lost the gene encoding the GLP-1 class of receptors, and likely the incretin action of GLP-1. Receptors that bind GLP-1, but yield glucagon-like action, have been characterized in goldfish and zebrafish, and these sequences are most closely related to glucagon receptors. Both pufferfish and zebrafish have a second glucagon receptor-like gene that is most closely related to the characterized goldfish glucagon receptor. The phylogeny of glucagon receptor-like genes in fish indicates that a duplication of the glucagon receptor gene occurred on the ancestral fish lineage, and could explain the shared action of glucagon and GLP-1. We suggest that the binding specificity of one of the duplicated glucagon receptors has diverged, yielding receptors for GLP-1 and glucagon, but that ancestral downstream signaling has been maintained, resulting in both receptors retaining glucagon-stimulated downstream effects.     

Glucagon-like peptide 1 immunoreactivity in gastroentero-pancreatic endocrine tumors: a light- and electron-microscopic study

The preproglucagon gene encodes, in addition to glucagon, two smaller peptides with structural similarity: glucagon-like peptides 1 and 2. Glucagon-like peptide 1 (GLP-1) 7–36 amide is the most powerful incretin candidate. In the present study, GLP-1 immunoreactivity was investigated in tissue specimens of various types of gastroenteropancreatic tumors, and the serum-levels of GLP-1 were assayed. Immunohistochemical staining of 88 tumors revealed GLP-1 immunoreactivity in 17 neoplasias (19.3 %), viz., in 7 out of 33 non-functioning tumors, 4 out of 20 gastrinomas, 4 out of 13 insulinomas, 1 out of 3 vasoactive-intestinal-polypeptide (VIP)omas and 1 adrenocorticotropic-hormone (ACTH)-producing tumor. In these tumors, GLP-1-immunoreactive cells were distributed either diffusely, arranged in clusters, or as single cells. All GLP-1-positive tumors were immunoreactive for glucagon or glicentin, 10 tumors were immunoreactive for pancreatic polypeptide, and 8 tumors for insulin. Ultrastructural analysis of 8 GLP-1-positive tumors, with the immunogold technique, demonstrated GLP-1 immunoreactivity mainly in cells resembling the A-cells of the pancreas or the L-cells of the gut. Of the 17 GLP-1-immunoreactive tumors, 15 were primarily located in the pancreas. Additionally, 2 non-functioning tumors of the rectum were GLP-1 immunoreactive. Five tumors were GLP-1 immunoreactive from 9 patients with multiple endocrine neoplasia I syndrome. Patients with GLP-1-immunoreactive tumors were characterized by a significantly lower rate of distant metastases (P<0.01) and a higher rate of curative resections (P<0.05). In 2 out of 22 patients, elevated serum-levels of GLP-1 were found: one patient with a vasoactive-intestinal-polypeptide (VIP)oma and 1 patient with a non-functioning tumor. This indicates that GLP-1 might be secreted at least by a few gastroenteropancreatic endocrine tumors.     

Glucagon-like peptide-1(7-37) does not stimulate either hepatic glycogenolysis or ketogenesis

Recent Studies have demonstrated that glucagon-like peptide-1 (GLP)(7-37) has more potent insulinotropic activity than glucagon. We therefore examined the effect of GLP-1(7-37) on liver metabolism using rat liver perfusion system. Ten nM GLP-1(7-37) did not affect glucose, ketone body and cAMP outputs from the perfused liver. Whereas, the same dose of glucagon stimulated these outputs significantly. When 10 nM GLP-1(7-37) perfused 5 min before the administration of 10 nM glucagon, the above stimulatory effects of glucagon were not affected. These results indicate that truncated GLP-1 has no effect on hepatic glycogenolysis and ketogenesis dissociating from its potent insulinotropic activity.     

Effect of truncated glucagon-like peptide 1 on cAMP in rat gastric glands and HGT-1 human gastric cancer cells

We tested the truncated 7-37 glucagon-like peptide 1 (TGLP-1), a naturally occurring porcine intestinal peptide, and other members of the glucagon family, including pancreatic glucagon (G-29), GLP-1 and GLP-2 for their ability to activate the cAMP generating system in rat gastric glands and HGT-1 human gastric cancer cells. In rat fundic glands, TGLP-1 was about 100 times more potent (EC50 = 2.8 X 10(-9) M) than GLP-1 of G-29, and 10 times more potent than G-29 in the HGT-1 cell line. Our results support the notion that TGLP-1 plays a direct role in the regulation of acid secretion in rat and human gastric mucosa.     

Identification and characterization of glucagon-like peptide-1 7-36 amide-binding sites in the rat brain and lung

High-affinity binding sites for glucagon-like peptide-1 7-36 amide (GLP-1 7-36 NH2) were identified in rat brain and lung membranes. Binding of [125I]GLP-1 7-36 NH2 was rapid, reversible, specific, saturable and pH dependent. Specific binding in the central nervous system was particularly high in the hypothalamus and the brain stem. Oxyntomodulin, glucagon-like peptide-1, glucagon-like peptide-2 and glucagon were 100-1000-fold less potent than GLP-1 7-36 NH2 in competition for this binding site.