Endocrine cells in the ileum of patients with irritable bowel syndrome.

AIM: To study the ileal endocrine cell types in irritable bowel syndrome (IBS) patients. METHODS: Ninety-eight patients with IBS (77 females and 21 males; mean age 35 years, range 18-66 years) were included, of which 35 patients had diarrhea (IBS-D), 31 patients had a mixture of both diarrhea and constipation (IBS-M), and 32 patients had constipation (IBS-C) as the predominant symptoms. The controls were 38 subjects (26 females and 12 males; mean age 40 years, range 18-65 years) who had submitted to colonoscopy for the following reasons: gastrointestinal bleeding, where the source of bleeding was identified as hemorrhoids (n = 24) or angiodysplasia (n = 3), and health worries resulting from a relative being diagnosed with colon carcinoma (n = 11). The patients were asked to complete the: Birmingham IBS symptom questionnaire. Ileal biopsy specimens from all subjects were immunostained using the avidin-biotin-complex method for serotonin, peptide YY (PYY), pancreatic polypeptide (PP), enteroglucagon, and somatostatin cells. The cell densities were quantified by computerized image analysis, using Olympus cellSens imaging software. RESULTS: The gender and age distributions did not differ significantly between the patients and the controls (P = 0.27 and P = 0.18, respectively). The total score of Birmingham IBS symptom questionnaire was 21 ± 0.8, and the three underlying dimensions: pain, diarrhea, and constipation were 7.2 ± 0.4, 6.6 ± 0.4, and 7.2 ± 0.4, respectively. The density of serotonin cells in the ileum was 40.6 ± 3.6 cells/mm² in the controls, and 11.5 ± 1.2, 10.7 ± 5.6, 10.0 ± 1.9, and 13.9 ± 1.4 cells/mm² in the all IBS patients (IBS-total), IBS-D, IBS-M, and IBS-C patients, respectively. The density in the controls differed significantly from those in the IBS-total, IBS-D, IBS-M, and IBS-C groups (P < 0.0001, P = 0.0001, P = 0.0001, and P < 0.0001, respectively). There was a significant inverse correlation between the serotonin cell density and the pain dimension of Birmingham IBS symptom questionnaire (r = -0.6, P = 0.0002). The density of PYY cells was 26.7 ± 1.6 cells/mm(2) in the controls, and 33.1 ± 1.4, 27.5 ± 1.4, 34.1 ± 2.5, and 41.7 ± 3.1 cells/mm² in the IBS-total, IBS-D, IBS-M, and IBS-C patients, respectively. This density differed significantly between patients with IBS-total and IBS-C and the controls (P = 0.03 and < 0.0001, respectively), but not between controls and, IBS-D, and IBS-M patients (P = 0.8, and P = 0.1, respectively). The density of PYY cells correlated significantly with the degree of constipation as recorded by the Birmingham IBS symptom questionnaire (r = 0.6, P = 0.0002). There were few PP-, enteroglucagon-, and somatostatin-immunoreactive cells in the biopsy material examined, which made it impossible to reliably quantify these cells. CONCLUSION: The decrease of ileal serotonin cells is associated with the visceral hypersensitivity seen in all IBS subtypes. The increased density of PYY cells in IBS-C might contribute to the constipation experienced by these patients.

Increased intracellular content of enteroglucagon in proximal colon is related to intestinal adaptation to germ-free status.

Changes in the frequency of endocrine cells are evidence of intestinal adaptation to germ-free (GF) status. Not only the distribution of these cells along the intestine, but also the differences in intracellular content of these regulatory peptides may be explored to explain functional and structural aspects of GF intestinal adaptation. Focusing on the endocrine L-cells, we analyzed the intracellular content of enteroglucagon (EG) and peptide YY (PYY) throughout the intestine of the 14 GF and 14 conventional (CV) mice by using immunohistochemistry and the supra-optimal dilution technique. The percentage of EG-immunoreactive cells, but not of PYY-immunoreactive cells stained at supra-optimal dilution was significantly higher in the proximal colon of GF mice than in the CV counterparts (P < 0.05). Since the content of co-stored PYY did not differ between GF and CV mice, the higher content of EG was compatible with a selective cellular response. Moreover, in the cecum of GF mice, the density of EG-immunoreactive cells was significantly higher than that of PYY-immunoreactive cells (P < 0.05). These results are consistent with preferential production of EG by L-cells at the expense of PYY in the proximal colon and in the enlarged cecum of GF mice. In addition, they may reflect the dynamics of the GF intestinal epithelium and/or be correlated with the higher serum levels of these peptides. The role of endocrine cells needs to be better studied in human and other experimental adaptative conditions in order to elucidate the regulatory mechanisms of intestinal functions.

Human rectal endocrine cells and aging.

Endocrine cells of the human rectum were investigated by immunocytochemistry and quantified by computerized image analysis in three different age groups. The age intervals were 20-29, 40-49 and 60-69 years. No statistically significant differences were found between the age groups, regarding the numbers of all endocrine cell types investigated, namely peptide YY (PYY)-, pancreatic polypeptide (PP)-, enteroglucagon-, somatostatin- and serotonin-immunoreactive cells. Nor was there any difference regarding the cell secretory index. Nuclear volume was significantly greater in the 40-49 year olds than the other age groups. There was no statistically significant difference between females and males regarding numbers of the endocrine cell types investigated. It is concluded that age does not affect the endocrine cells of the human large intestine as it was earlier found in animal models of aging. It is imperative that caution should be taken when applying results obtained in animal models of aging in humans.

Large intestinal endocrine cells in non-obese diabetic mice.

Colonic endocrine cells from prediabetic and diabetic non-obese diabetic mice as well as of the sister strain, BALB/cJ, were investigated by immunocytochemistry and computer image analysis. In prediabetic mice, enteroglucagon-and serotonin-immunoreactive cells were significantly increased in number, whereas the cell secretory index of these two cell types was significantly reduced. No significant differences were found in numbers or cell secretory index of peptide YY (PYY)-immunoreactive cells. In diabetic mice, PYY-immunoreactive cells were significantly fewer, but there were no significant differences in the numbers of enteroglucagon-and serotonin-immunoreactive cells. Whereas the cell secretory index was reduced in serotonin-producing cells, no significant differences were found between diabetic and control mice regarding the cell secretory index of PYY- and enteroglucagon-immunoreactive cells. Nor was any statistically significant difference found between controls, prediabetic and diabetic non-obese diabetic mice, regarding the thickness of submucosa, of circular and longitudinal-muscle layers, or of the mucosal area/microm baseline. The present study showed that abnormalities in colonic endocrine cells do occur, in both prediabetic and diabetic mice, but they are different in nature and can be divided into primary and secondary to the diabetes onset. The present findings of abnormal colonic endocrine cells in non-obese diabetic mice, an animal model for human insulin-dependent diabetes mellitus, might help explain the gastrointestinal disorders observed in patients with diabetes. The study also showed that the change in the colonic endocrine cells is dynamic and started before the onset of the diabetic condition.

Distribution of enteroglucagon- and peptide YY-immunoreactive cells in the intestinal mucosa of germ-free and conventional mice.

There are evidences that microflora modulates endocrine cells in the gastrointestinal tract. In the present study we investigated the distribution of EG- and PYY-immunoreactive cells throughout the intestine of adult male NMRI conventional and germ-free mice. EG-immunoreactive cells were significantly more frequent in the proximal and middle colon than in the remainder of the intestine in both groups. In germ-free animals, these cells were more frequent in the cecum and less frequent in the distal ileum compared to conventional mice. PYY-immunoreactive cells were more frequent in the distal colon than in the remainder of the intestine in both groups, but they were significantly more frequent in the middle and distal colon of germ-free animals than in that of conventional counterparts. The number of EG-immunoreactive cells was 4.5-fold higher than the number of PYY-immunoreactive cells in the cecum of germ-free mice. The present results indicate the existence of an inverse gradient of EG- and PYY-immunoreactive cells along the colon, which is not significantly changed in the absence of a microflora. PYY production seems to be more significant in the distal colon. The cecum and the proximal portion of the colon are probably the regions of greatest functional importance for EG production, which is related to the microflora and probably to fermentation products, whether or not the effect of this peptide is trophic or antitrophic.

Complex co-localization of chromogranins and neurohormones in the human gastrointestinal tract.

Co-localization of chromogranin (Cg) A, B, and C has been studied in different neuroendocrine cell types in histologically normal mucosa from human gastrointestinal tract (corpus, antrum, duodenum, ileum, and colon) using single-, double-, and triple-immunofluorescence stainings. Virtually all enterochromaffin (EC) cells contained CgA, and those in the luminal two thirds of the antral mucosa and villi of small intestine often also contained CgB. A few EC cells in the duodenal crypts contained CgC. Most gastrin cells harbored both CgB and CgA, although rather more CgB than CgA, but some gastrin cells contained all three types, i.e., also CgC. Some CCK cells also contained all three chromogranins. Enteroglucagon cells in the duodenal villi contained CgA and some CgB. CgA (but not B or C) was found in some secretin, GIP, enteroglucagon/peptide YY, and neurotensin cells. A few somatostatin cells contained CgA but neither CgB nor CgC. CgA and C were found mainly in the basal cell region, whereas CgB occurred more diffusely throughout the cytoplasm. This varying distribution suggests that not all secretory granules contain CgA, or that CgB may occur in a nongranular form. The varying composition of the different chromogranins may reflect their complex functional roles in the widespread neuroendocrine system.

Immunocytochemical detection of islet hormones in the digestive system of Protopterus annectens.

The presence, distribution, and interrelationships of the four typical pancreatic islet hormones were investigated in the digestive system of Protopterus annectens by single and double immunohistochemical methods. Insulin-, glucagon-, and somatostatin-immunoreactive (IR) elements were detected in both the pancreas and the gut. Pancreatic polypeptide (PP)-IR endocrine cells were always present in the gut, but were only present in the pancreas of a few specimens. Some of the latter cells also seemed to react with glucagon antiserum. In the pancreas the immunopositive cells were organized into islets of different sizes, and their organizations were studied by the double immunohistochemical techniques. In the few large islets insulin-IR cells were present in the central zone, glucagon- and PP-IR cells at the periphery, and somatostatin-IR cells intermingled with both the peripheral and the central endocrine cells. In the smaller islets, the number and the staining intensity of glucagon- and PP-IR endocrine cells varied markedly. In the gut, insulin-, somatostatin-, and PP-IR cells were of the open type; glucagon-containing cells were very few and had no luminal contact. They were differently distributed along the intestinal epithelium. Somatostatin-IR nerve fibers and somatostatin-IR neuron cell bodies were also observed in the intestinal wall. The organization of pancreatic endocrine cells in P. annectens is similar to that observed in the majority of teleosts even if a different topographical association can be found. Furthermore, islets of different sizes seem to display a different metabolic turnover, and the detection of pancreatic PP-immunoreactivity varied according to the specimens utilized. In the intestinal portion insulin-IR cells, in addition to PP-, glucagon- and somatostatin-IR cells are present: this suggests that intestinal insulin-like immunoreactivity may be more widespread than previously supposed.

Endocrine cells in rectal biopsy specimens from patients with familial amyloidotic polyneuropathy.

BACKGROUND: A decreased amount of duodenal endocrine cells has recently been found in patients with familial amyloidotic polyneuropathy (FAP), and it has been suggested that this may contribute to the development of the gastrointestinal symptoms. The present study was performed to establish whether the endocrine cells in the lower gastrointestinal tract are also affected. METHODS: The endocrine cells in rectal biopsy specimens from 13 patients (7 women and 6 men) with FAP were investigated by means of immunocytochemistry and computed image analysis. These specimens were taken early during the disease. As controls, rectal specimens from 13 patients (7 women and 6 men) with rectal bleeding caused by haemorrhoids or polyps were included. RESULTS: The amount of both serotonin- and pancreatic polypeptide (PP)-immunoreactive cells was significantly decreased compared with the controls. There was no significant difference between patients and controls with regard to the amount of chromogranin A-, polypeptide YY (PYY)-, enteroglucagon- and somatostatin-immunoreactive cells. CONCLUSION: It has been suggested that serotonin depletion may contribute to the development of the severe constipation encountered in FAP patients in early stages of the disease. This constipation may promote bacterial overgrowth in the small intestine, with diarrhoea and malabsorption as a result.

Immunological detection of prohormone convertases in two different proglucagon processing cell lines.

The distribution of the prohormone convertases, PC1/3, PC2 and PC5/6, was determined by immunoblotting in two cell lines. In alpha TC1-6 cells, the proglucagon processing occurred according to the pancreatic A-cell type. In STC-1 cells, proglucagon was processed in a manner reminiscent of the intestinal L-cell type. PC1/3 was undetectable in both proglucagon processing cell lines whereas PC2 displayed a strong immunostaining in the alpha TC1-6 cells and was barely detectable in the STC-1 cells. PC5/6 was detected as a 70 kDa protein in both cell lines. These results suggest a possible role of PC2 in the processing of proglucagon into glucagon in the A-cells, whereas in L-cells it would require still undetermined endoproteases.

Distribution and immunocytochemical colocalization of peptide YY and enteroglucagon in endocrine cells of the rabbit colon.

Peptide YY (PYY) is 36 amino acid peptide hormone present in high concentrations in the colon where it is colocalized with enteroglucagon in L cells. A selective release of PYY and enteroglucagon from the rabbit colon has been described, raising the question of the exact localization of the two hormones in the rabbit colon. We have therefore examined the distribution of PYY and enteroglucagon as well as somatostatin in the rabbit colon using RIA and electron microscopic immunocytochemistry. PYY and enteroglucagon were present in high concentrations in the colorectal mucosa with peak concentrations in the left colon (PYY 544 +/- 87 pmol/g, enteroglucagon 152 +/- 10 pmol/g). Electron microscopic examination of the colonic mucosa demonstrated a large population (65%) of EC cells, a moderate population (30%) of L cells, and a small population (5%) of D cells. By immunogold labeling serotonin was localized to EC cells, PYY and enteroglucagon to L cells, and somatostatin to the D cell. Double immunogold labeling revealed PYY and enteroglucagon in all L cells examined (93 cells). A majority of the secretory granules (83%) were labeled by both PYY and glucagon antibodies, whereas a significant portion of granules (15%) was labeled by the PYY antibodies alone. The results demonstrate that L cells are the sole source of PYY and enteroglucagon in the rabbit colon and that L cells contain different populations of secretory granules. The existence of different secretory granules in L cells may explain the selective release of PYY and enteroglucagon observed in the rabbit colon.

Rat small intestinal morphology and tissue regulatory peptides: effects of high dietary fat.

Sprague-Dawley rats (3 weeks old) were fed on isoenergetic diets in which 40% of the total energy was provided as fat either in the form of butter (high saturated fat), olive oil (high monounsaturated fat) or maize oil (high polyunsaturated fat), with one group on low-fat (10% of total energy) standard diet as a control. Animals were killed after 8.4 (se 0.8) weeks by cardiac puncture. Similar pieces of jejunum and ileum were prepared for morphometric studies. Extracts of tissue from the proximal and distal segments of the whole small intestine from four animals per group were assayed using established techniques for enteroglucagon, motilin, neurotensin, somatostatin, substance P and vasoactive intestinal peptide (VIP). We found that maize oil and olive oil increased villus height: crypt depth ratio in both jejunum and ileum. Maize oil increased tissue concentrations of somatostatin (P less than 0.05) and substance P (P less than 0.005) in the proximal segment. Both maize oil and olive oil increased tissue concentrations of neurotensin and substance P (P less than 0.005) in the distal segments. These observations may explain the improvement of intestinal absorption of fluid following supplementation with polyunsaturated fat.

Immunocytochemical study of endocrine cells in pelvic ileal reservoirs.

The distribution and morphology of intestinal endocrine cells was investigated in the mucosa of pelvic ileal reservoirs using immunocytochemical methods. Endoscopic biopsies were obtained from 15 patients after the construction of a modified J-pouch. The mucosa of the reservoir showed a variable degree of colonic metaplasia in all cases. No relevant quantitative variations of gut endocrine cells were detected, as revealed by immunostaining for the general marker, chromogranin, compared with normal ileal mucosa. Immunostaining for different peptide-containing cells resulted in normal number and morphology of serotonin, enteroglucagon, peptide tyrosine-tyrosine, and somatostatin-containing cells. Neurotensin cells were less numerous than in normal mucosa. The role played by gastrointestinal hormones in the adaptive response of the intestine to pouch construction is, presently, unclear. Further studies involving measurements of fasting and meal-stimulated levels of gut hormones in pouch patients might clarify this aspect.

Molecular and cellular analysis of a neoplastic pancreatic A cell tumor.

Endocrine tumors of the pancreas may produce characteristic syndromes attributable to the increased secretion of one or more hormones. These tumors provide valuable opportunities for the analysis of hormone biosynthesis and secretion in the neoplastic human endocrine cell. The authors studied a pancreatic endocrine tumor obtained from a patient with classical glucagonoma syndrome. Characterization of plasma and tumor glucagon-like immunoreactivity (GLI) by high-performance liquid chromatography and radioimmunoassay for GLI showed different chromatographic profiles, with glucagon the major molecular form in the tumor, and glicentin and oxyntomodulin predominating in plasma. Although immunocytochemical staining of the tumor showed only focal weak positivity for glucagon, tumor extracts contained large amounts of immunoreactive GLI peptide. Northern blot analysis of tumor RNA demonstrated that abundant glucagon mRNA transcripts were present, just slightly larger in size than those detected in normal pancreas and intestine. Electron microscopic analysis of the tumor cellular ultrastructure revealed only occasional small electron dense secretory granules. A large number of complex lysosome-like structures of variable size and electron density were detected throughout the cytoplasm and ringing the nucleus of most cells, a feature atypical of endocrine tumors of the pancreas. Primary cultures of dispersed tumor cells were established and, in contrast to previous results, were obtained using normal or neoplastic islet cell models, GLI secretion was found to be stimulated eightfold by incubation with 5 mM dibutyryl cyclic adenosine monophosphate. Phorbol myristate acetate, the calcium ionophore A23187, and sodium butyrate had no effect on GLI secretion in vitro. These observations indicate that neoplastic human A cells may have abnormalities at different points in the biosynthesis and secretion of glucagon.

1H n.m.r. conformational studies on the C-terminal octapeptide of oxyntomodulin, a beta-turn locked by a salt bridge.

The octapeptide Lys-Arg-Asn-Lys-Asn-Asn-Ile-Ala (Arg4 in the human sequence) is the C-terminal part of porcine oxyntomodulin, an endogeneous peptide which is a potent inhibitor of stimulated acid secretion. This octapeptide exhibits the whole range of biological activities of the parent hormone. In the present work we report an 1H n.m.r. investigation of the conformational properties of the octapeptides of pig and human sequences in dimethylsulfoxide-d6 (DMSO) solution. The various resonances were assigned on the basis of two-dimensional COSY and NOESY experiments. Other experiments such as (i) temperature and concentration dependence of the amide proton chemical shifts, (ii) effects of ionic strength, (iii) comparison of the spectra with different analogues, were performed. We showed that in DMSO, the conformation of the octapeptide is directly related to the ionisation state of the C-terminus carboxyl group of alanine. In carboxylic state, the peptide adopts an extended conformation, while in the carboxylate state the four last residues (Asn-Asn-Ile-Ala) are involved in a type II beta-turn structure probably locked by a salt bridge between the carboxyl group of Ala8 and the epsilon ammonium group of Lys4 (or the guanidinium group of Arg4). These observations provide an insight into the possible conformational tendencies of this peptide in biological media.

Heterotopic gastric mucosa of the rectum--characterization of endocrine and mucin-producing cells by immunocytochemistry and lectin histochemistry. Report of a case.

Heterotopic gastric mucosa in the rectum is particularly uncommon; only 23 cases have been reported to date. Moreover, no studies have been done on the neuroendocrine apparatus and glycoprotein production of the heterotopic mucosa. This study reports on a 13-year-old boy, admitted with rectal bleeding and persistent tenesmus. An ulcerative lesion was found on colonoscopy; biopsies revealed a fundic-type gastric tissue. Medical therapy (H2-blockers) promptly healed the rectal ulcer; surgical excision of the heterotopia was performed with complete and permanent relief of symptoms (3-year follow-up). Immunocytochemistry (PAP) revealed 5-Ht and somatostatin cells in the gastric-type mucosa, as in the normal human stomach. These cells also were present in the surrounding rectal epithelium where PYY-enteroglucagon cells were detected, which were absent in the heterotopic tissue. Mucin histochemistry showed PAS-positive cells also strongly stained by LA lectin in the heterotopic tissue, differentiating the rectal epithelium that remained unstained. Therefore, the morphofunctional status (endocrine cells and mucins) of the gastric heterotopia was almost identical to its orthotopic counterpart, confirming the hypothesis that endocrine cells and mucin-producing cells differentiate their metabolic products according to the anatomic and functional activity of the epithelium where they grow.

Oxyntomodulin and glicentin: brain-gut peptides in the rat.

Glucagon-like materials and glucagon have been identified by immunoassay and immunocytochemistry in the mammalian central nervous system. However, the molecular forms relevant to brain glucagon-like immunoreactivity (GLI) have not been precisely defined. In the rat small intestine, more than 90% of GLI is constituted by two peptides: oxyntomodulin (OXM) and glicentin. This work was initiated to characterize and determine the concentrations of these two peptides and glucagon in the rat central nervous system and to compare their relative proportions with those found in the gut. Different regions from the adult rat brain were analyzed by HPLC in association with RIA, using a central glucagon antiserum and an antibody directed toward the C-terminal end of OXM and glicentin. The elution profiles of hypothalamus extracts were constituted by two main peaks, both detected by the two antibodies used and displaying the same retention times as glicentin and OXM, respectively. A third small peak, which coeluted with glucagon, was constantly recorded with the central glucagon antiserum. The percentages of glicentin, OXM, and glucagon in 10 hypothalami were 37 +/- 1%, 55 +/- 1%, and 8 +/- 2%, respectively (n = 8). This distribution was quite similar to that in small intestinal extracts (38 +/- 1%, 59 +/- 1%, and 1.3 +/- 0.1%, respectively; n = 7); however, the peptide concentrations were almost 50-fold greater in intestine than in hypothalamus. In the medulla oblongata, the same peptide ratio was observed, with 10-fold lower concentrations compared to those in hypothalamus. In olfactory bulb, cerebellum, and cortex the concentrations were close the the detection limit, whereas they could be not detected in the pituitary. The combination of HPLC and specific RIAs allowed us to unambiguously characterize OXM and glicentin as the major components of GLI in the rat hypothalamus and medulla oblongata. The same proportion of these two peptides in the central nervous system and the gut indicates that a similar posttranslational processing exists in these rat tissues, another example of the brain-gut axis.

Chronic pancreatitis and diabetes mellitus: plasma and gastroduodenal mucosal profiles of regulatory peptides (gastrin, motilin, secretin, cholecystokinin, gastric inhibitory polypeptide, somatostatin, VIP, substance P, pancreatic polypeptide, glucagon, enteroglucagon, neurotensin).

A disturbed intraduodenal milieu and pancreatic scarring in advanced chronic pancreatitis (CP) may lead to changes of gut and pancreatic hormones. In the present study, the gastroduodenal mucosal content of several regulatory peptides was determined in 8 patients with severe calcific CP and 8 healthy volunteers. In addition, hormone release into the bloodstream was estimated after intraduodenal acid/glucose stimulation in the control subjects and 8 CP patients each with or without secondary diabetes mellitus (DM), and in 8 patients with juvenile DM, so that disturbed gut hormone release could be attributed either to CP or DM. While VIP release into the circulation was similar in all participants, mucosal levels of VIP and substance P were significantly elevated in the duodenal bulb and descending duodenum of CP patients. The somatostatin content of gastroduodenal mucosa in CP was at least as high as in normals. Gastrin was significantly more abundant only in the duodenal bulb of CP patients, while plasma gastrin was normal. Duodenal CCK concentrations tended to be elevated in the duodenal bulb, but not significantly. The release of secretin seemed to be higher in type-1 diabetics than in CP patients. The mucosal pattern of GIP was nearly identical in CP patients and controls. Compatible with this finding, the GIP release did not show any peculiarities in CP with or without DM or in DM. Basal and stimulated plasma levels of motilin were abnormally high in CP. Pancreatic polypeptide plasma levels were normal in DM, but significantly reduced in CP, especially in CP with DM. Fasting PP and stimulated pancreatic enzyme outputs were linearly related.(ABSTRACT TRUNCATED AT 250 WORDS)

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

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 immunoreactivites 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.

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.

Distribution of oxyntomodulin and glucagon in the gastrointestinal tract and the plasma of the rat.

Oxyntomodulin (OXM), an intestinal glucagon-containing peptide extended at its C-terminal end by an octa-peptide, is one of the gut glucagon-like immunoreactants (GLI) or enteroglucagon. The distribution of OXM and glucagon was determined in the gastrointestinal tract and in the plasma of the rat. Reversed-phase HPLC, associated with RRA or RIA, performed with an N-terminally directed glucagon antiserum (GOL), was used. HPLC of intestinal extracts or plasma separated the GOL immunoreactivity into three peaks: two major peaks coeluting with a preparation of rat glicentin (peak I, partially purified from rat intestine) and porcine or rat OXM, respectively, and a smaller peak coeluting with glucagon. The behavior of the three peaks in the analytical systems matched that of glicentin, OXM, and glucagon, respectively, allowing their identification. The concentrations of OXM picomoles per g of tissue) gradually increased from the duodenum (9 +/- 1) to ileum (93 +/- 4), thereafter decreasing in cecum and colon (22 +/- 3). In the gut, OXM, glucagon, and peak I averaged 40%, 1%, and 59% of the total GLI, respectively. OXM was present in significant amounts in the pancreas (18% of GLI) and stomach (27% of GLI), two tissues in which it accounted, together with glucagon, for almost the totality of GLI. In 24 h-fasted rats, plasma concentrations of OXM, glucagon, and peak I, determined after HPLC with GOL antiserum, were 15.1 pM, 8.6 pM, and 12.3 pM, respectively. Two hours after refeeding, both OXM and peak I were significantly increased (P less than 0.05 and P less than 0.02) by a similar factor (2-fold), while glucagon remained unchanged. When the HPLC results were compared with RIA measurement of GLI (GOL antiserum) and glucagon (with a C-terminal glucagon antiserum) in plasma, enteroglucagon (GOL--C-terminal glucagon antiserum immunoreactivities) correlated well with the sum of OXM plus peak I. The combination of HPLC and RRA or RIA allows the unambiguous determination of OXM, glucagon, and glicentin (peak I) in tissues and plasma. In the rat intestine and in the plasma, OXM and glicentin appear roughly in the same ratio and seem to be the major components, if not the totality, of enteroglucagon.