Prosomatostatin 1-64 is a major product of somatostatin gene expression in pancreas and gut.

Prosomatostatin (pro-SS) is a peptide of 92 amino acids which contains the extensively studied somatostatin (SS) 1-28 and SS 1-14 at the C terminus. Little is known about the N-terminal part of pro-SS. In previous studies, using a radioimmunoassay against pro-SS 20-36 (sequence deduced from human cDNA sequence) we have identified a peptide with a molecular mass of approximately 8000 daltons in extracts of pancreas and intestinal mucosa. Using a variety of chromatographic procedures we have now isolated this peptide from extracts of pancreas and intestinal mucosa from pigs. The isolated peptides were sequenced on an Applied Biosystems gas phase sequenator and cleaved with the Asp-N endopeptidase for sequencing of C-terminal fragments. The peptides had an amino acid sequence identical to human pro-SS 1-64. In effluent from isolated perfused preparations of porcine small intestine and pancreas we identified upon appropriate stimulation pro-SS 20-36 immunoreactive peptides that by isocratic high pressure liquid chromatography appeared identical to pro-SS 1-64. An identical peptide was identified in pig plasma. Thus, in pancreas and gut pro-SS processing gives rise to the same pro-SS 1-64 molecule in spite of differential processing of the C terminus (SS 1-14 in pancreas and SS 1-28 in gut). The eventual hormonal role of pro-SS 1-64 may now be evaluated.

Oxyntomodulin: a potential hormone from the distal gut. Pharmacokinetics and effects on gastric acid and insulin secretion in man.

Synthetic oxyntomodulin, a predicted product of the glucagon gene, which is produced in the human lower intestinal mucosa, was infused in doses of 100 and 400 ng kg-1 h-1 into six volunteers to study its pharmacokinetics and effects on pentagastrin-stimulated gastric acid secretion (100 ng kg-1 h-1). The concentration of oxyntomodulin in plasma measured with a cross-reacting glucagon assay increased from 37 +/- 5 to 106 +/- 17 and 301 +/- 40 pmol l-1, respectively. The metabolic clearance rate was 5.2 +/- 0.7 ml kg-1 min-1 and the half-life in plasma was 12 +/- 1 min. Oxyntomodulin reduced the pentagastrin-stimulated acid secretion by 20 +/- 9% during the low-rate infusion (P less than 0.05) and by 76 +/- 10% during the high-rate infusion (P less than 0.05). In accordance with the homology with glucagon, there was a small, significant rise in plasma concentrations of insulin and insulin C-peptide during oxyntomodulin infusion. Oxyntomodulin may therefore be included among the potential incretins and enterogastrones in man.

Oxyntomodulin (glicentin-(33-69)): pharmacokinetics, binding to liver cell membranes, effects on isolated perfused pig pancreas, and secretion from isolated perfused lower small intestine of pigs.

The pharmacokinetics of purified synthetic oxyntomodulin were studied after infusing it into euglycaemic pigs at two rates. The elimination of the peptide from plasma was characterized by two components, a fast one (t1/2 7.2 +/- 0.6 min) and a slow one (t1/2 20.4 +/- 3.8 min) (mean +/- S.E.M., n = 7). The metabolic clearance rate was independent of infusion rate (6.96 +/- 0.99 vs 7.44 +/- 0.98 ml/kg . min (mean +/- S.E.M., n = 7). The synthetic peptide bound to pig hepatic glucagon receptors, but with approximately 2% of the affinity of glucagon, and showed insulinotropic and somatostatinotropic effects when infused into isolated perfused pig pancreases at concentrations higher than 10(-10) M. A dose-dependent increase was also shown for pancreatic glucagon output. A naturally occurring peptide, identified as oxyntomodulin by gel filtration and HPLC, was released into the circulation from the pig lower small intestinal mucosa upon intraluminal administration of glucose, and represented 25 +/- 3.8% of the secreted glucagon-like immunoreactivity. 11 +/- 2.3% of the secreted glucagon-like immunoreactivity was indistinguishable from glucagon itself upon gel filtration; thus at least 36% of the glucagon-like immunoreactivity secreted from the intestinal mucosa is already in an active form.

Processing and secretion of prosomatostatin by the pig pancreas.

Antisera and radioimmunoassays against five different regions of prosomatostatin (proSS) were used for chromatographical analysis and for immunohistochemical mapping of the products of proSS in the pig pancreas. Secreted products of proSS were studied by analysis of effluent from isolated perfused pig pancreas obtained during isoproterenol stimulation. All cells that were stained with one antiserum also stained with the other antisera. Immunoreactive nerves were not observed. Isoproterenol increased equally the secretion of proSS 20-36, proSS 65-76, and proSS 79-92 immunoreactivity. The major molecular forms identified in pancreatic extracts and released from the pancreas were proSS 79-92; proSS 65-76; an N-terminally extended form of proSS 65-76; and two larger forms comprising the proSS 20-36 sequence (but not the 1-13 sequence) with and without the proSS 65-76 sequence. ProSS 1-10, 1-32 and 65-92 (somatostatin 28) were not identified.

Localization in the gastrointestinal tract of immunoreactive prosomatostatin.

Antisera against 5 different regions of the entire prosomatostatin molecule were used for immunohistochemical mapping of prosomatostatin-containing structures in the pig gastrointestinal tract, and for radioimmunological and chromatographical analysis of the products of prosomatostatin in extracts of ileal mucosa. The latter showed that the antisera were capable of identifying components containing N-terminal as well as C-terminal parts of prosomatostatin. Endocrine cells were identified with all antisera in most parts of the gastrointestinal tract, and varicose nerve fibres were observed in all parts of the small intestine but not in the stomach and the colon. The colon contained very few immunoreactive structures. Immunoreactive nerve cell bodies were found in the submucous plexus of the small intestine. All immunoreactive endocrine cells in the stomach and the duodenum and all immunoreactive nerves were stained by all 5 antisera whereas the small intestinal endocrine cells did not stain for the most N-terminal region of prosomatostatin. The results suggest that all gastrointestinal somatostatin is derived from the same precursor molecule, which, however, in the small intestinal endocrine cells is processed differently from that of the other tissues.

Gastrin-releasing peptide in the porcine pancreas.

The presence of gastrin-releasing peptide (GRP) was studied in extracts of porcine pancreata. Gel filtration and high-pressure liquid chromatographic profiles of these extracts as monitored with both C-terminally and N-terminally directed radioimmunoassays against GRP showed pancreatic GRP to consist of one main form, namely the 27-amino acid peptide originally extracted from porcine stomach, and small amounts of a C-terminal fragment identical with the C-terminal 10-amino acid peptide. Gastrin-releasing peptide-like immunoreactivity released from the isolated perfused porcine pancreas during electrical vagal stimulation was shown by gel filtration to consist of the same two forms. By use of immunocytochemical techniques employing an antiserum directed against its N terminus, GRP was localized to varicose nerve fibers in close association with the exocrine tissue of the porcine pancreas in particular. Some fibers were found penetrating into pancreatic islets also. Immunoreactive nerve cell bodies as well as fibers were found within intrapancreatic ganglia. The potency of GRP in stimulating exocrine as well as endocrine secretion from the porcine pancreas, its presence in close contact with both acini and islets, and its release during vagal stimulation indicate that GRP may have a role in the parasympathetic regulation of endocrine and exocrine secretion from the pig pancreas.

Glucagon-like peptides GLP-1 and GLP-2, predicted products of the glucagon gene, are secreted separately from pig small intestine but not pancreas.

We developed specific antibodies and RIAs for glucagon-like peptides 1 and 2 (GLP-1 and GLP-2), two predicted products of the glucagon gene, and studied the occurrence, nature, and secretion of immunoreactive GLP-1 and GLP-2 in pig pancreas and small intestine. Immunoreactive GLP-1 and GLP-2 were identified in glucagon-producing cells of the pancreatic islets, and in glicentin-producing cells of the small intestine. Immunoreactive GLP-1 and 2 in intestinal extracts corresponded in molecular size to peptides synthesized according to the predicted structure. By reverse phase HPLC, intestinal and synthetic GLP-1 behaved similarly, whereas synthetic and intestinal GLP-2 differed. Pancreatic extracts contained a large peptide with both GLP-1 and GLP-2 immunoreactivity. Secretion was studied using isolated perfused pig pancreas during arginine stimulation, and isolated perfused pig ileum during either luminal glucose stimulation or vascular administration of the neuropeptide, gastrin-releasing peptide (GRP). Immunoreactive GLP-1 and GLP-2 were secreted in parallel with pancreatic glucagon and intestinal glicentin. The molecular forms of secreted immunoreactive GLP-1 and 2 corresponded to those identified in the tissue extracts.

Glicentin 1-61 probably represents a major fraction of glucagon-related peptides in plasma of anaesthetized uraemic pigs.

Uraemia was induced in pigs by ligation of the renal vascular pedicle, and uraemic plasma was analysed for glucagon and glucagon-related peptides. A preponderance of large molecular weight (Mr) components comprising glicentin and moieties of slightly lower Mr was found, accounting for 73 +/- 3% (mean +/- SEM, n = 12) of the total plasma glucagon-like immunoreactivity. Comparisons with glicentin 1-61, produced by controlled, stepwise, consecutive digestion of purified natural glicentin with carboxypeptidases (carboxypeptidase A followed by carboxypeptidase B, and again by carboxypeptidase A and B), gel filtration, ion exchange chromatography, reverse phase HPLC and radioimmunoassays for the glucagon sequences 6-15 and 19-29 and for the glicentin sequence 12-30 all indicate that glicentin 1-61 constitutes approximately 57% of the large Mr glucagon-related peptides found in uraemia in pigs.

The intestinal mucosa preferentially releases somatostatin-28 in pigs.

We studied the molecular forms of somatostatin-like immunoreactivity (SLI), newly released from isolated perfused preparations of the porcine antrum, stomach, pancreas and upper small intestine: Perfusion effluents were concentrated by Sep-Pak C-18 adsorption, eluted with ethanol, dessicated, and subjected to gel filtration with subsequent radioimmunoassays for somatostatin-14 and N-terminal somatostatin-28 immunoreactivity. All the SLI newly released from the stomach and antrum eluted at the position of somatostatin-14, and such was also the case for more than 95% of the SLI newly released from the pancreas, while 68 -/+ 7% and 75 -/+ 8% of the SLI newly released from the isolated perfused jejunum and ileum, respectively, corresponded to somatostatin-28. By reverse phase HPLC the identity of these peptides with synhetic somatostatin-14 and -28 was established.

Mouse salivary glands secrete a glucagon-degrading enzyme, not glucagon.

Salivary glands have been reported to synthetize glucagon in various animal species. We therefore studied the glucagon-like immunoreactivity (GLI) in mouse saliva. Stimulation with phenylephrine evoked a 15-fold increase of salivary GLI output. On Sephadex G-50 gel filtration, the salivary GLI was significantly larger than glicentin, the hitherto largest known glucagon-related peptide; furthermore, the immunoreactivity was not absorbable on glucagon immunoadsorbent. 125I-Labeled glucagon incubated with high GLI containing saliva, and subjected to gel filtration and immunoadsorption was degraded to low molecular weight, nonimmunoreactive moieties. Among EDTA, phenylmethylsulfonylfluoride, Pepstatin-A, and N-ethylmaleimide, only N-ethylmaleimide inhibited the degradation. Renin, also found in mouse saliva, degraded the tracer but did not cochromatograph with salivary GLI. In conclusion, GLI of mouse saliva is not a peptide containing a glucagon-immunoreactive sequence but represents tracer-degrading activity, probably composed of sulfhydryl enzymes.

Distribution and molecular forms of peptides containing somatostatin immunodeterminants in extracts from the entire gastrointestinal tract of man and pig.

Specimens from human porcine mucosal and muscular tissue from the entire gastrointestinal tract were extracted in acid ethanol, subjected to chromatography and analysed for somatostatin-like immunoreactivity by region-specific radioimmunoassays. The concentration of somatostatin-like immunoreactivity from man and pig ranged from 1.13 +/- 0.37 to 101.15 +/- 33.93 pmol/g wet weight, and from 7.64 to 159.48 +/- 23.79 pmol/g wet weight, respectively. In both species the highest concentrations were found in the jejunum. The immunoreactivity in intestinal mucosal extracts was distributed among four major peaks, two of which were identified by HPLC as somatostatin 1-28 and somatostatin 1-14, respectively. A peak of approx. 10 kDa was resolved by ion exchange plus HPLC into three components, two containing at least part of the somatostatin 1-14 sequence as well as (part of) the somatostatin 1-28(1-14) sequence (but differing in charge), the third containing only the 1-28(1-14) sequence. These peptides probably represent uncleaved and partially cleaved prosomatostatin. The fourth component to be identified by gel filtration was slightly larger than somatostatin 1-14. Extracts from the antrum, the pancreas and from muscular tissues contained almost exclusively somatostatin 1-14, and very little somatostatin 1-28, indicating that the somatostatin precursor is processed differently at these sites. Furthermore, extracts of porcine gastric antrum, analysed for somatostatin 1-28(1-14) immunoreactivity, showed two immunoreactive forms in the mucosa and three major forms in the muscular layers.

Glicentin is present in the pig pancreas.

Specimens from porcine pancreas and ileal mucosa were extracted in acid/ethanol, subjected to gel permeation chromatography, ion-exchange chromatography, enzymatic peptide degradation, reverse-phase HPLC, and analysed for glucagon-like and glicentin-like immunoreactivity by region-specific radioimmunoassays. Results obtained with all methods were consistent with the hypothesis that glicentin is present in the pig pancreas in small amounts.

On the contamination of commercially available aprotinins with pancreatic islet peptides.

Contamination of aprotinins with immunoreactive insulin, glicentin, glucagon, somatostatin, pancreatic polypeptide and pancreatic polypeptide-icosapeptide was estimated by radioimmunoassay. High somatostatin concentrations were found in two commercial preparations. Influence of somatostatin contamination of aprotinins on the clinical as well as the laboratory applications of aprotinin may thus be anticipated.

Glucagon-related peptides in the human gastrointestinal mucosa.

We studied the chromatographic profile and the distribution of glucagon-related peptides in the human gastrointestinal mucosa, using radioimmunoassays directed against the glucagon 6-15 and 19-29 sequences, and against the glicentin sequences 15-30 and 61-69, and a radioreceptor assay for glucagon. Very small amounts of glucagon-related peptides were found in the gastric mucosa, whereas at least four different components could be identified in the distal intestine. One component (mean concentration 130 pmol/g ileal mucosa) is similar to porcine glicentin for size and C-terminal extension, but differs from the glucagon part of the molecule in the N-terminal extension. A second component (mean concentration 131 pmol/g) is probably identical to porcine peak II enteroglucagon (glicentin 33-69), and a third component (7.9 pmol/g) seems to be identical with glucagon. A fourth component containing the glucagon sequence plus an N-terminal extension was also identified (1.7 pmol/g). Thus the human intestinal mucosa contains large amounts of peptides containing the glucagon sequence; at least one of these probably also possesses glucagon-like bioactivity. The proposed structures of the four components are consistent with the base sequence of the first half of the human glucagon gene.

Somatostatin 1-28 circulates in human plasma.

The gel filtration profile of immunoreactive somatostatin in human plasma in the fasting state is not well established as a consequence of insufficient sensitivity of the combined chromatography and radioimmunoassay procedures usually employed. We here report the gel filtration profiles of plasma samples after somatostatin concentration by batchwise immunoaffinity chromatography. The results clearly and reliably document the presence of a circulating peptide in human plasma with a gel permeation chromatography profile identical to the one of synthetic somatostatin 1-28. Approximately 46% of the total somatostatin-like immunoreactivity in plasma is due to this component.