Isolation and chemical characterization of glicentin C-terminal hexapeptide in porcine pancreas

Using a radioimmunoassay specific for porcine glicentin C-terminal hexapeptide, we isolated a peptide from porcine pancreas and characterized it as the C-terminal 64-69 sequence of glicentin: H-Asn-Lys-Asn-Asn-Ile-Ala-OH. The purification steps included gel filtration, ion-exchange chromatography and HPLC. In each step, the recovery of the desired peptide, radioimmunologically estimated from the respective elution profile, was 71.4-91.7%. The final yield of the hexapeptide was 22 micrograms (4.3%) from 800 g pancreas. The pancreatic content of this peptide was estimated to be approximately equimolar to that of pancreatic glucagon. No hexapeptide-like component was detected in porcine intestinal extracts. The data confirmed that the processing of pancreatic proglucagon liberates the C-terminal hexapeptide of the intramolecular glicentin sequence in a tissue-specific manner during the production of glucagon.     

Specific inactivation of lysosomal glycosidases in living fibroblasts by the corresponding glycosylmethyl-p-nitrophenyltriazenes.

Glicentin (a highly purified 100-amino acid peptide with glucagon-like immunoreactivity from porcine gut) was subjected to limited digestion with trypsin and carboxypeptidase B, and the resulting peptides were studied by gel filtration and region-specific glucagon radioimmunoassays. Similar digests of glucagon and purified fragments of glucagon were studied in parallel. Glicentin gave rise to peptides that corresponded closely to the 1-17 and 19-29 fragments of glucagon. Also, 125I-labelled glicentin and 125I-labelled glucagon gave rise to identical fragments after trypsin treatment. On the basis of this and other evidence [Jacobsen, Demandt, Moody & Sundby (1977) Biochim. Biophys. Acta 493, 452-459] it is concluded that glicentin contains the entire glucagon sequence at residues number 64-92 and thus fulfills one of the requirements for being a 'proglucagon'.     

The primary structure of porcine glicentin (proglucagon)

The primary structure of porcine glicentin has been established. The molecule consists of 69 amino acid residues and has a molecular weight of 8128. The sequence of glicentin 1–30 represents the glicentin-related pancreatic peptide (GRPP) previously isolated from porcine pancreas. The sequence 33–61 represents the full sequence of glucagon and the sequence 64–69 is a C-terminal hexapeptide. These three sequences, GRPP, glucagon and the hexapeptide are linked by two Lys-Arg pairs which probably represent the sites for post-synthetic enzymatic cleavages. Glicentin thus fulfils the structural requirements for being proglucagon.     

Effect of highly purified porcine gut glucagon-like immunoreactivity (glicentin) on glucose release from isolated rat hepatocytes

We studied the effect of the highly purified gut peptide glicentin on the glucose production and cyclic AMP accumulation of isolated rat hepatocytes. Glicentin at 2.10(-7) mol/l had the same effect on glucose production as maximally effective concentrations of glucagon, but did not stimulate cyclic AMP to the same extent; furthermore, glicentin apparently had only 1/100 of the potency of glucagon on glucose production. During incubation with hepatocytes glicentin was degraded to low molecular weight fragments one of which were chromatographically very similar to fragments of glucagon. It is suggested that glicentin exerts its glucagon-like effects on hepatocytes only after degradation to glucagon-like fragments. The results also demonstrate that the coupling between cyclic AMP accumulation and glucose production depends on the nature of the stimulatory peptide.     

Coexistence of peptide YY and glicentin immunoreactivity in endocrine cells of the gut

Endocrine cells containing peptide YY (PYY) were numerous in the rectum, colon and ileum and few in the duodenum and jejunum of rat, pig and man. No immunoreactive cells could be detected in the pancreas and stomach. Coexistence of PYY and glicentin was revealed by sequential staining of the same section and by staining consecutive semi-thin sections. Since the PYY sequence is not contained in the glucagon/glicentin precursor molecule the results suggest that the PYY cell in the gut expresses two different genes coding for regulatory peptides of two different families.     

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.     

Glucagon-like immunoreactivity in hypothalamic neurons of the rat

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

Co-existence of glicentin and peptide YY in colorectal L-cells in cat and man. An electron microscopic study

Electron microscopic immunocytochemistry using protein A-gold labelling of ultrathin sections revealed immunoreactive glicentin (gut-type glucagon) and peptide YY (PYY) in virtually all secretory granules in a population of L-type endocrine cells in feline colon and human rectum. The granules of the human glicentin/PYY cells were considerably smaller in size than those in the cat. In both species the results indicate co-existence of glicentin and PYY in the same secretory granules, despite the probable derivation of the two peptides from two different precursors.     

Immunohistochemical studies on glucagon, glicentin and pancreatic polypeptide in human stomach: normal and pathological conditions

Summary Endocrine-like cells containing glucagon, glicentin or pancreatic polypeptide immunoreactivity in human foetal and adult stomach, with or without disease, were studied with the indirect immunoperoxidase method and mirror sectioning technique. In foetal and neonatal oxyntic mucosae, there were endocrine-like cells with glucagon and glicentin immunoreactivities and argyrophilia. Cells containing glicentin immunoreactivity alone were detected earlier than glucagon cells during foetal development, and were also distributed throughout foetal to neonatal life. Bovine pancreatic polypeptide immunoreactivity coexisted in a subpopulation of the glucagon-glicentin cells. These cells were absent from normal oxyntic mucosa in the postneonatal period and from normal antral mucosa throughout life. Hamartomatous polyp in adult oxyntic mucosa, hyperplastic oxyntic mucosa in Menetrier's disease and atrophic oxyntic mucosa in a remnant stomach with cancer showed scattered glucagon-glicentin cells, but few or no cells containing bovine pancreatic polypeptide. Intestinalized mucosa showed plentiful glicentin cells with occasional glucagon and/or bovine pancreatic polypeptide immunoreactivity. Some gastric cancer cells of both diffuse and adenoplastic types contained immunoreactive glicentin and, less frequently, glucagon. Bovine pancreatic polypeptide immunoreactivity was detected in a few adenoplastic cancer cells, but not in diffuse type cells. Three different anti-pancreatic polypeptide sera against bovine, porcine or human pancreatic polypeptide detected basically the same cells mentioned above, but pancreatic polypeptide cells lacking human pancreatic polypeptide immunoreactivity were also present in foetal oxyntic mucosa. Immunoabsorption tests revealed that the bovine pancreatic polypeptide immunoreactivity was remote from peptide YY and neuropeptide Y.     

Development of an oxyntomodulin/glicentin C-terminal radioimmunoassay using a "thiol-maleoyl" coupling method for preparing the immunogen

Oxyntomodulin (OXM) and glicentin, two peptides processed from proglucagon, both contain the glucagon sequence and a C-terminal basic octapeptide, KRNRNNIA extension. A method to produce antibodies, directed specifically toward the C-terminal extension of these two peptides, was developed; it consisted of the use of thioled bovine serum albumin conjugated with the synthetic N-maleoyl C-terminal octapeptide as the immunogen. Three rabbits (FAN, LEG, and PIP) generated antisera with affinity constants close to 5 X 10(10) M-1. In the radioimmunoassay system, these antisera showed a 100% cross-reactivity with OXM, partially purified rat and human glicentin, and the C-terminal 19-37 OXM fragment. They displayed no cross-reactivity toward the glucagon molecule. The cross-reactivity of C-terminal fragments of OXM demonstrated that the epitope involves the C-terminal hexapeptide and that the two last amino acid residues are essential for the binding. The high-performance liquid chromatography elution profiles of human jejunum or rat intestinal extracts obtained by radioimmunoassay with LEG antiserum showed two major peaks which had the same retention times as OXM and glicentin markers. Thus, the major end products in the human and rat small intestine are OXM and glicentin. In human or rat pancreas, the two main peaks detected were glucagon and the C-terminal hexapeptide of OXM/glicentin. Small amounts of OXM were also found in pancreas, whereas no significant quantities of glicentin could be detected. The "thiol-maleoyl" coupling method described here, and applied to produce C-terminal OXM/glicentin specific antisera, might be of general use to obtain antibodies against a well-defined epitope.     

Glicentin: A precursor of glucagon?

The relationships between glucagon and gut-glucagon like immunoreactants (gut-GLIs) have been investigated by immunofluorescence in canine gut mucosa. The R64 antiserum, raised against the purified gut-GLI-l glicentin, and which does not react with porcine glucagon, revealed immunofluorescent cells in the gastric and intestinal mucosa. Glicentin positive cells of the stomach oxyntic glands were also stained by N- and C- terminally directed antiglucagon sera, corresponding to the gastric A-cell. In the small and large intestine, glicentin immunoreactive cells reacted solely with the cross-reacting (N-terminal) glucagon antiserum, belonging to the L-cells. Based on chemical and immunochemical data, it has been suggested that glicentin could represent an intermediate in the glucagon biosynthesis. Therefore, the results of this immunofluorescence study, showing glicentin and glucagon immunodeterminants in the A-cell, strongly support such an hypothesis. In addition the presence of glicentin like material in the A- and L-cells suggests that these two cell types synthesize their secretory product via a common precursor.     

Cell-specific post-translational processing of preproglucagon expressed from a metallothionein-glucagon fusion gene

Glucagon is a peptide hormone of 29 amino acids encoded by a preprohormone which contains in tandem the sequences of glucagon and two additional glucagon-like peptides (GLPs) structurally related to glucagon and separated by intervening peptides. Glucagon arises by cleavage from the prohormone within the A cells of the pancreatic islets but in the intestine remains as part of a partially processed precursor (glicentin). To determine whether additional glucagon-like peptides are processed from preproglucagon and to analyze for potential cellular specificity in the processing of preproglucagon, we introduced and expressed a metallothionein-glucagon fusion gene in a fibroblast and two endocrine (pituitary and pancreatic islet) cell lines. Chromatographic analyses of cell extracts utilizing specific radioimmunoassays to chemically synthesized peptides demonstrate the liberation of intact glucagon, glicentin, GLP-I(1-37), GLP-I(7-37), GLP-II, and an intervening peptide amidated at its carboxyl terminus. The peptides were present in distinct yet different patterns in the two endocrine but not the fibroblast cell lines. The cell-specific liberation of the glucagon-like and intervening peptides suggests their potential as new bioactive peptides. The cellular specificity in the processing of preproglucagon indicates that the genetic determinants of the processing activity are complex and are expressed in a cell-specific manner.     

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.     

Evidence that enteroglucagon (II) is identical with the C-terminal sequence (residues 33-69) of glicentin.

Enteroglucagon (II) was isolated from extracts of pig ileum mucosa by repeated gel filtrations, and its immunochemical and chromatographic characteristics were compared with those of a synthetic peptide corresponding to the 33-69 sequence of pig glicentin, before and after digestion with trypsin or trypsin followed by carboxypeptidase B, by using five region-specific assays covering most of the glicentin sequence. Enteroglucagon (II) and the synthetic peptide behave identically under three different conditions of chromatography as determined with all five assays (including a highly specific radioreceptor assay), and gave rise to similar fragments after enzyme digestion. It was therefore concluded that enteroglucagon (II) and the 33-69 sequence of glicentin are most probably identical.     

Complete sequences of glucagon-like peptide-1 from human and pig small intestine

In the small intestine, proglucagon is processed into the previously characterized peptide "glicentin" (proglucagon (PG) 1-69) and two smaller peptides showing about 50% homology with glucagon: glucagon-like peptide-1 and -2. It was assumed that the sites of post-translational cleavage in the small intestine of the proglucagon precursor were determined by pairs of basic amino acid residues flanking the two peptides. Earlier studies have shown that synthetic glucagon-like peptide-1 (GLP-1) synthesized according to the proposed structure (proglucagon 71-108 or because residue 108 is Gly, 72-107 amide) had no physiological effects, whereas a truncated from of GLP-1, corresponding to proglucagon 78-107 amide, strongly stimulated insulin secretion and depressed glucagon secretion. To determine the amino acid sequence of the naturally occurring peptide we isolated GLP-1 from human small intestine by hydrophobic, gel permeation, and reverse-phase high performance liquid chromatography. By analysis of composition and sequence it was determined that the peptide corresponded to PG 78-107. By mass spectrometry the molecular mass was determined to be 3295, corresponding to PG 78-107 amide. Furthermore, mass spectrometry of the methyl-esterified peptide showed an increase in mass compatible with the presence of alpha-carboxyl amidation. Thus, the gut-derived insulinotrophic hormone GLP-1 is shown to be PG 78-107 amide.     

Immunocytochemical localization of insulin- and glucagonlike peptides in rat salivary glands

An avidin-biotin immunocytochemical technique was used to localize cells containing an insulin- or glucagon-like peptide in the major salivary glands of Sprague-Dawley rats. Cells with insulin-like staining were observed in the intercalated ducts of both the parotid and submandibular glands, but none were found in the sublingual gland. A discrete population of cells with intense glucagon-like immunostaining was associated with the acini of all three major salivary glands. This immunostaining only followed use of a glucagon antiserum with N-terminal specificity and not after incubation of tissues with an anti-glucagon serum having C-terminal specificity. These results suggest that rat salivary glands may contain peptides potentially capable of influencing substrate metabolism. In addition, the present findings indicate that the glucagon-like peptide found in salivary glands has a greater immunocytochemical similarity to glicentin (gut-type glucagon) and/or glucagon precursors than to the 3500 molecular weight pancreatic glucagon.     

Immunoreactive glucagons purified from dog pancreas, stomach and ileum

Previous studies have shown that pig intestine contains a 69 amino acid glucagon (glicentin) as well as a 37 amino acid glucagon (oxyntomodulin). In pig pancreas the 29 amino acid glucagon predominates. Since glucagon is thought to be expressed from a single gene in mammals, these differences in molecular forms indicate differential posttranslational processing of the glucagon precursor by different tissues. In the current study glucagon immunoreactivity (IR) was separately purified from dog pancreas, stomach mucosa and ileum mucosa. Purification and sequence analysis of the different tissue glucagons show that dog pancreas and stomach mucosa contain glucagon-29 while ileum mucosa contains glucagon-37 and glucagon-69. The latter is the major form present with glucagon-37 accounting for only 10-20% of the total ileum glucagon content. The N-terminal 32 amino acid portion of dog glucagon-69 differs at 6 sites from pig glucagon-69: RSLQDTEEKSRSFSAPQTEPLNDLDQMNEDKR... The C-terminal glucagon-37 is identical to pig oxyntomodulin.     

Two Dipolar α-Helices within Hormone-encoding Regions of Proglucagon Are Sorting Signals to the Regulated Secretory Pathway

Proglucagon is expressed in pancreatic α cells, intestinal L cells, and some hypothalamic and brainstem neurons. Tissue-specific processing of proglucagon yields three major peptide hormones as follows: glucagon in the α cells and glucagon-like peptides (GLP)-1 and -2 in the L cells and neurons. Efficient sorting and packaging into the secretory granules of the regulated secretory pathway in each cell type are required for nutrient-regulated secretion of these proglucagon-derived peptides. Our previous work suggested that proglucagon is directed into granules by intrinsic sorting signals after initial processing to glicentin and major proglucagon fragment (McGirr, R., Guizzetti, L., and Dhanvantari, S. (2013) J. Endocrinol. 217, 229–240), leading to the hypothesis that sorting signals may be present in multiple domains. In the present study, we show that the α-helices within glucagon and GLP-1, but not GLP-2, act as sorting signals by efficiently directing a heterologous secretory protein to the regulated secretory pathway. Biophysical characterization of these peptides revealed that glucagon and GLP-1 each encode a nonamphipathic, dipolar α-helix, whereas the helix in GLP-2 is not dipolar. Surprisingly, glicentin and major proglucagon fragment were sorted with different efficiencies, thus providing evidence that proglucagon is first sorted to granules prior to processing. In contrast to many other prohormones in which sorting is directed by ordered prodomains, the sorting determinants of proglucagon lie within the ordered hormone domains of glucagon and GLP-1, illustrating that each prohormone has its own sorting “signature.”     

Ontogeny of immunoreactive glicentin in the human gastrointestinal tract and endocrine pancreas

The gestational time of appearance and distribution of immunoreactive glicentin was compared to that of immunoreactive glucagon in the gastrointestinal tract and endocrine pancreas of human fetuses, aged between 5 and 24 weeks, by an indirect immunoperoxidase method. With the glicentin antiserum No. R 64, the first immunoreactive cells were detected at the 10th week of gestation in the oxyntic mucosa and proximal small intestine, at the 8th week in the ileum and at the 12th week in the colon. In the endocrine pancreas, the first immunoreactive cells were observed as early as 8 weeks within the walls of the primitive pancreatic ductules. At a more advanced stage of development (12 weeks), they were found interspersed among the islet cell clusters and still later (16 weeks) inside the recognizable islets of Langerhans. With the glucagon antiserum No. GB 5667, no immunoreactive cells were demonstrated in the gastrointestinal tract whatever the age of the fetuses. In the endocrine pancreas, the first immunoreactive cells were observed at the 8th week of gestation in the pancreatic parenchyma. The distribution of glucagon-containing cells in the pancreas was similar to that of glicentin immunoreactivity throughout ontogenesis. In the pancreatic islets of one 18-week-old human fetus, the study of consecutive semithin sections treated by both antisera showed that the same cells were labelled. The significance of these findings concerning the role of glicentin as a glucagon precursor is discussed.     

Distribution of glucagon-like peptide I in canine and feline pancreas and gastrointestinal tract

Recently, a putative hormone, glucagon-like peptide I (GLP I), has been identified in the predicted sequences of the precursors to pancreatic glucagon in human, rat, hamster, and ox. The distribution of GLP I immunoreactivity in canine and feline pancreas and gastrointestinal tract was examined immunohistochemically and was compared with that of two other antigenic determinants of pancreatic pro-glucagon, i.e., glucagon and the NH2 terminus of glicentin. All three determinants occurred in the same population of islet cells in normal pancreas and in pancreas consisting predominantly of islet tissue from dogs with canine pancreatic acinar atrophy. Northern blot analysis of mRNA from the latter tissue, using a rat pre-pro-glucagon complementary DNA probe, revealed a single mRNA species similar in size to the pre-pro-glucagon mRNA detected in fetal rat pancreas. The three antigenic determinants of pancreatic pro-glucagon were co-localized also in intestinal L-cells and in canine gastric A-cells. Canine and feline pancreatic pro-glucagons therefore resemble those identified in other mammals and may also occur in gastrointestinal endocrine cells. Although there is evidence that the GLP I sequence is not liberated from pancreatic pro-glucagon, our results raise the possibility that this putative hormone may be a cleavage product of pro-glucagon in the gastrointestinal tract.