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Postpartum mothers and their healthcare providers often face the challenge of limited data regarding the safety of drug therapies during lactation. Pregnancy can lead to sustained weight gain, and obesity can negatively impact both physical and psychological well-being. The introduction of GLP-1 agonists to augment weight loss has become a topic of interest for many postpartum mothers. Our study aims to investigate the transmission of semaglutide into human milk in the first steps to ensure the safety and health of both lactating mothers and their breastfed infants. Semaglutide quantification was performed using high-resolution liquid chromatography-mass spectrometry. InfantRisk Center Human Milk biorepository released milk samples from eight women collected at 0, 12 and 24 h post-semaglutide administration. Semaglutide was extracted using protein precipitation in methanol, followed by chromatographic separation. Linear calibration curves for the method ranged between 2.5-30 ng/mL, with a limit of detection of 1.7 ng/mL and a limit of quantification of 5.7 ng/mL (LLOQ). Semaglutide was not detected in any of the collected human milk samples. A worst-case scenario of the relative infant dose (RID) was calculated using the LLOQ as the drug concentration in milk when considering semaglutide's bioavailability and long-acting dose profile. The maximum RID projected was 1.26%, far below the standard 10% safety threshold. While questions about long-term infant outcomes, the safety of maternal nutrient intake, and the nutrient content of breast milk remain, our findings suggest that semaglutide concentrations in human milk are unlikely to pose clinical concerns for breastfed infants. These results support healthcare providers in making informed decisions regarding postpartum therapeutic interventions.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The bioactive fraction of "enteroglucagon" has been isolated from the porcine jejuno-ileum on the basis of its glucagon-like action in liver. High performance liquid chromatography analysis followed by Dansylation of peptide fragments obtained after enzymatic digestion of both this peptide and glucagon suggests that the bioactive "enteroglucagon" contains the glucagon molecule plus a C-terminal extension under the from of the octapeptide. Lys-Arg-Asn-Lys-Asn-Asn-Ile-Ala-COOH. The presence in the native molecular of a structural difference of modification as compared to glucagon in the 1-11 fragment is possible.

The effect of total parenteral nutrition (TPN) in rats on a number of enteroendocrine cells was investigated. The rats were given a continuous intravenous infusion of basal TPN solution for 7 days. Samples from duodenum, jejunum and ileum were collected, immunostained and the immunoreactive cells quantified using a computerised morphometrics system. The endocrine cells containing somatostatin, cholecystokinin (CCK), gastric inhibitory polypeptide (GIP), neurotensin and enteroglucagon were investigated. The results demonstrated a significant reduction in the number of CCK cells in the duodenum and jejunum. In the ileum the neurotensin-immunoreactive cells were significantly increased in number (P less than 0.02). No change was seen in the number of cells immunostained for somatostatin, GIP or enteroglucagon. These data indicate that short term TPN has a definite effect on the enteroendocrine cell population which may be linked to the side effects of TPN seen in man.

1. Analytical subcellular fractionation techniques have been applied to endoscopic human rectal biopsies to study the localization of enteroglucagon, somatostatin, vasoactive intestinal peptide and the properties of the principal subcellular organelles. 2. The peptide hormones, detected by radioimmunoassay, showed particulate localizations with single peaks in the density gradients for enteroglucagon (modal density 1.25) and somatostatin (modal density 1.23). Vasoactive intestinal peptide showed a less discrete localization but demonstrated a major peak (modal density, 1.17) with a small subsidiary peak (modal density 1.24). 3. The following organelles, characterized by their marker enzymes, were located in the density gradients; plasma membrane (5'-nucleotidase), mitochondria (malate dehydrogenase), peroxisomes (catalase), lysosomes (beta-N-acetyl-D-glucosaminidase), endoplasmic reticulum (neutral alpha-D-glucosidase) and cytosol (lactate dehydrogenase). 4. This technique can be used to investigate disease of the human rectum at a subcellular level.

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.

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.

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.

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)

Correlative immunocytochemical and electron microscopic studies, using the semi thin-thin technic, were performed to identify the (entero) glucagon, somatostatin and pancreatic polypeptide-like immunoreactive cells of the human colonic mucosa. Mean granule diameter for each cell type was estimated according to two methods and histograms showing the granule size distribution were constructed. A total of 139 immunostained cells identified at the ultrastructural level were analyzed. Mean granule diameter for (entero)glucagon-containing cells was 318 +/- 11 nm but a reduction of granule size with age was noteworthy: granules were larger in the fetus (mean diameter 350 +/- 15) than in adults (mean diameter 310 +/- 10 nm). Somatostatin-containing cells, very rare in adults, were present in the fetal distal colon. Their general mean granule diameter was 354 +/- 18 nm but many cells had a mean granule diameter of more than 400 nm. A pancreatic polypeptide-like immunoreactivity was found only in (entero)glucagon-containing cells, pointing out the possible occurrence of both peptides (or of similar sequences) in the same cells. Previous ultrastructural studies dealing with a tentative classification of the human colonic endocrine cells were compared with the present data.

Gut endocrine cells in a total of 122 intestinal-tract adenocarcinomas induced in inbred Wistar rats by 1,2-dimethylhydrazine dihydrochloride were examined histologically, ultrastructurally, and immunohistochemically for gastrin, somatostatin, vasoactive-intestinal polypeptide (VIP), and glicentin (enteroglucagon). Of the 122 tumors, argyrophil cells were detected in 42 tumors (34.3%) comprising 15 tumors of the well differentiated type and 27 tumors of the poorly differentiated type, including signet-ring-cell carcinomas. Of the 27 tumors of the poorly differentiated type, 12 were regarded as endocrine-cell carcinomas composed of numerous argyrophil or argentaffin cells and mucus-containing cells. Immunohistochemically, 7 of the 12 tumors had glicentin and two of these seven tumors also had gastrin and argentaffin cells synchronously. None of the tumors showed immunoreactivity for somatostatin and VIP. Nine of the 12 tumors metastasized to the lung, pancreas, liver, mesenterium, omentum, and lymph nodes. The metastatic foci of these tumors were also shown to have glicentin and argentaffin cells. Ultrastructurally, four types of endocrine granule were found in the tumor cells and amphicrine cells containing endocrine granules and mucous granules were noted. These endocrine-cell tumors were assumed to develop from totipotent immature cells of endodermal origin.

We describe here a depletion of peptide containing nerves and cells in Hirschsprung's disease, in comparison with specimens of bowel taken from age-matched neonates with no evidence of chronic constipation. VIP content in the diseased specimens was reduced by almost 80%, from 110/+-10.6 (mean +/- SEM) pmol VIP/g wet weight of tissue in controls to 23.8 +/- 3.5 pmol/g in the mid-portion of the diseased specimens. In addition, the numbers of enteroglucagon and somatostatin cells in the mucosa were significantly reduced in the aganglionic portions. Enteroglucagon cells were reduced from 55 +/- 7 in controls to 27 +/- 2 in proximal portions rising to 44 +/- 3 and 49 +/- 4 cells/mm2 in middle and distal areas. Somatostatin cell numbers also fell, from 5.5 +/- 1.9 to 1.8 +/- 0.8, 2.5 +/- 0.7 and 3.8 +/- 0.9 cells/mm2 in similar areas. Further investigation of the abnormalities of the diffuse neuroendocrine system in Hirschsprung's disease may help in understanding the nature of this condition and provide additional information on the role of these peptides in the control of gut function.

Reliable and specific radioimmunoassays have been developed for the gut hormones secretin, gastrin, cholecystokinin, pancreatic glucagon, VIP, GIP, motilin, and enteroglucagon. Using these assays, the relative pattern of distribution of the gut hormones has been determined using the same bowel extracts for all measurements. VIP occurred in high concentration in all regions of the bowel, whereas secretin, GIP, motilin, and CCK were predominantly localised in the proximal small intestine. Pancreatic glucagon was almost exclusively confined to the pancreas. Like VIP, enteroglucagon also exhibited a wide pattern of distribution but was maximal in the ileum. The acid ethanol extraction method that was used was found to be unsuitable for gastrin. On gel chromatography of the extracts, motilin and VIP eluted as single molecular species in identical position to the pure porcine peptides. CCK, pancreatic glucagon, enteroglucagon and GIP were all multiform.

The changes occurring in the ileal wall and in enteroglucagon cells were studied in a rat model of intestinal adaptation, obtained by the transposition of a segment of distal ileum into the mid-duodenum (6 rats, compared with 6 transected controls). After 40 days, the transposed ileal segment, compared to the equivalent segment in the controls, showed striking increase in weight, especially of the epithelium (1,585 +/- 127 vs. 305 +/- 42 mg, mean +/- SEM, p less than 0.0005). The calculated weight of enteroglucagon cells in the segment showed a smaller, but significant increase (1.7 +/- 0.3 vs. 0.8 +/- 0.2 micrograms, p less than 0.05). Plasma enteroglucagon was markedly raised (239 +/- 28 vs. 61 +/- 7.1 pmol/l, p less than 0.0005) and showed a greatly increased meal-stimulated response (1,521 +/- 284 vs. 83 +/- 43 pmol, p less than 0.0005), thus suggesting hyperactivity of enteroglucagon cells.

The gastrointestinal tract is a large and complex endocrine organ. Elucidation of its nature, and the actions of its individual hormonal components, has advanced in recent years following the purification and sequencing of six probable hormones and the subsequent development of radioimmunoassay techniques for their measurement and immunocytochemistry for the determination of their cellular origin. Immunoassay and immunocytochemistry are clearly complimentary to each other in providing a fuller understanding of the dynamics of endocrine function at the cellular level. This is illustrated in the examples described in this paper.

Several segments of the gastrointestinal tract of the white-belly opossum Didelphis albiventris were investigated immunocytochemically for the occurrence of polypeptide YY (PYY) and enteroglucagon (GLU). PYY- and GLU-immunoreactive cells were observed in the lower part of the ileum, cecum and colon. These cells were seen to emit cytoplasmic basal processes to the neighbouring cells with a number of them reaching the glandular lumen via apical cytoplasmic process. GLU-immunoreactive cells were also present in the oxyntic mucosae and in the pancreatic duct. Staining of consecutive sections for the two polypeptides, respectively, revealed the coexistence of immunoreactivity for PYY and GLU in the same cell type.

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.

The morphology and histochemistry of gastroduodenal endocrine tumors from 16 patients were studied. All patients underwent operation, in most cases with a preoperative diagnosis of nonendocrine tumor, ulcer, o polyp(s). The argentaffin reaction was positive in three tumors, and the Hellerström--Hellman argyrophil reaction was positive in four tumors. All tumors reacted positively to the Grimelius argyrophil stain, and 13 were positive with the Sevier--Munger argyrophil stain. Gastrin immunoreactivity was found in eight tumors, and substance-P immunoreactivity in seven tumors. No enteroglucagon, adrenal cortex hormone, or pancreatic polypeptide was observed in any of the tumors. Three patients with Sevier--Munger-positive gastric tumors had concurrent pernicious anemia, and 2 patients with gastrin-immunoreactive tumors had acute or chronic gastroduodenal ulceration. The results indicate that the gastroduodenal endocrine tumor as a rule gives no endocrine symptoms and that the tumor type is an unexpected finding at operation. The tumors may contain gastrin, substance P, somatostatin, and serotonin and tend to be multihormonal.

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.

The mucosal concentrations of seven regulatory peptides and the density properties and integrity of their storage granules have been studied in mucosal biopsies from the human jejunum in eight gastrointestinal disease states and compared with normal controls. In diseases with associated mucosal inflammation (coeliac disease, Crohn's disease with jejunal involvement, postinfective tropical malabsorption, and common variable immunodeficiency) there was a selective increase in fragility of the gastric inhibitory polypeptide (GIP) and somatostatin storage granules. The gastrin, motilin, enteroglucagon, secretin, and vasoactive intestinal polypeptide granules had normal properties in these conditions. In diseases in which diarrhoea occurred in the absence of changes in jejunal mucosal histology (irritable bowel syndrome, pancreatic insufficiency, jejuno-ileal bypass for morbid obesity, and purgative abuse) there were no abnormalities of the storage granules. Increased mucosal concentrations of all peptides except vasoactive intestinal polypeptide (VIP) were found in coeliac disease and selective increases of VIP found in Crohn's disease, motilin in the irritable bowel syndrome and gastrin and GIP in pancreatic insufficiency. It is suggested that the storage granule abnormalities in the diseases with abnormal mucosal histology are secondary to the inflammatory changes.

The effect of jejunoileal bypass in lean and obese Zucker rats on a number of enteroendocrine cell types was investigated 5 weeks following surgery to remove 80 percent of the small bowel from continuity. The endocrine cells containing somatostatin, cholecystokinin, gastric inhibitory polypeptide, enteroglucagon and neurotensin were investigated. In control rats enteroglucagon cell number was decreased in obese compared to lean animals (5 +/- 1 vs 11 +/- 1 cells/mm). Following jejunoileal bypass the enteroglucagon cell population increased two-fold in both the functional and bypassed bowel in obese rats but was not elevated in lean animals. A significant increase in the number of cholecystokinin cells in the bypassed loop of the jejunum in both lean and obese bypassed rats was observed. The cholecystokinin cell population was also markedly elevated in the functional jejunum of obese but not lean bypassed rats. Only small changes were noted in cell numbers of gastric inhibitory polypeptide, somatostatin and neurotensin containing cells, suggesting that individual cell types have specific stimuli for proliferation. Epithelial height, a measure of intestinal adaptation, was similar in lean and obese rats in both the control and bypassed states, but weight loss in obese bypassed animals was significantly greater than that of lean bypassed rats. The hyperinsulinemia of obese rats was only partially normalized by jejunoileal bypass. These data indicate that jejunoileal bypass has effects on specific enteroendocrine cells which differ between lean and obese Zucker rats, and between individual cell types.(ABSTRACT TRUNCATED AT 250 WORDS)

Epithelial endocrine cells containing 5-hydroxytryptamine, substance P, somatostatin, enteroglucagon and vasoactive intestinal polypeptide-immunoreactivity were localized by immunocytochemistry in the mucosa of normal appendices, ileum and proximal colon, and in neurogenic appendicopathy. In neurogenic appendicopathy a large number of proliferating nerves were visualized independently of neurotransmitters by immunostaining for neuron-specific enolase. A large number of nerve fibers were shown to contain substance P-immunoreactivity and to be of intrinsic origin. Stromal endocrine cells containing 5-hydroxytryptamine, somatostatin- and possibly substance P-immunoreactivity, were observed in substantial numbers in neurogenic appendicopathy. Substance P may be involved as a neurotransmitter and/or as a paracrine/endocrine peptide in the pathogenesis of spastic contractions and abnormal peristalsis of the appendix, which are characteristic of neurogenic appendicopathy. Stromal endocrine cells may be considered to be the origin of certain carcinoids in the appendix.

In this study we have investigated the mucin profile and the endocrine cell population in gastric endoscopic biopsies from 22 patients affected by chronic gastritis and intestinal metaplasia and in five surgical specimens of stomachs removed because of intestinal-type carcinoma (4) or peptic ulcer (1). High iron diamine-Alcian blue (HID-Ab) staining and peptide immunocytochemistry (peroxidase anti-peroxidase technique) were used. Forty-one foci of intestinal metaplasia were detected, 15 produced sulphomucins and 26 sialomucins. Of the endocrine cells investigated, gastrin and somatostatin cells were the most frequently observed, while cholecystokinin, glucose-dependent insulinotropic peptide-, secretin- and enteroglucagon-containing cells were also found in the metaplastic areas, but less frequently. No significant correlation was found between the type of mucin and the types of endocrine cells present, the latter usually resembling those normally found in the small intestine. On the basis of these results we conclude that intestinal metaplasia involves mucin- and peptide-producing cells of the stomach in a variable manner, with no correlation between the two.

The localization and distribution of regulatory peptides was studied in separated epithelium, lamina propria, submucosa, and external muscular layer from 16 specimens of human bowel. Immunoreactive enteroglucagon, gastric inhibitory polypeptide, and neurotensin were almost confined to the epithelial fraction (97.5 +/- 2.2%, 97.5 +/- 4.2%, and 99.3 +/- 1.1% of their respective total content, mean +/- SEM) and were only localized in endocrine cells. Vasoactive intestinal polypeptide-, substance P-, and bombesinlike peptides were virtually restricted to the nonepithelial layers (99.6 +/- 0.2%, 99.6 +/- 0.2%, and 100%) and were demonstrated exclusively in nerves. A particularly rich vasoactive intestinal polypeptide- and substance P-immunoreactive nerve supply was seen in the nonepithelial mucosa, which contained the highest concentrations of these peptides, while bombesin was mainly recovered from the external muscle (87.7 +/- 2.7%). Somatostatin, measured with an antiserum highly specific for somatostatin-14, was found throughout the wall, mainly in the epithelium (39.9 +/- 5.2%) and lamina propria (29.5 +/- 5.9%), but could be immunostained only in endocrine cells.

This study was designed to investigate the mechanism of excessive weight loss following gastric surgery. Twelve weight-stable and six weight-losing postoperative subjects were studied. The weight-losing subjects had lesser body mass based upon anthropometric measurements. All weight-losing subjects and six weight-stable subjects excreted excess breath hydrogen after a standard meal. The quantities of hydrogen excreted by the weight-losing subjects and weight-stable subjects with positive tests were not different, implying similar degrees of carbohydrate malabsorption. Peak breath hydrogen concentration occurred earlier in weight-losing subjects than in weight-stable subjects (2 vs 5 hr), indicating more rapid oral-cecal transit of the test meal in weight-losing subjects. Analyses of postprandial blood samples for eight different gastrointestinal hormones demonstrated exaggerated postprandial elevations in the concentrations of enteroglucagon and neurotensin. The results of these studies demonstrate close correlations between excessive weight loss after gastric surgery, rapid gastrointestinal transit as measured by excess breath hydrogen excretion, and increased release of hormones from the distal intestine. We conclude that these hormones are secreted in excess due to the rapid gastrointestinal transit and that rapid gastrointestinal transit may play a role in excessive weight loss after gastric surgery.

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.

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.

"Gastric inhibitory peptide" or "glucose-dependent insulin-releasing peptide" (GIP) is a member of the gut hormone family. Its physiological action is thought to be related to its insulinotrophic effect. The occurrence and distribution of GIP was studied by immunohistochemistry. In all species examined including man, GIP immunoreactivity was found to reside in the glucagon cells of the pancrease and gut. Three pancreatic glucagonomas were found to contain numerous cells displaying GIP and glucagon immunoreactivity. The GIP antiserum used did not cross react with either pancreatic-type or gut-type glucagon (GLI).

A method for assaying specifically the biologically active peptides Glucagon-37 (G-37/Oxyntomodulin/bioactive Enteroglucagon) and Glucagon-29 (G-29/pancreatic Glucagon) has been developed by use of high performance liquid chromatography (HPLC) of crude tissue extracts followed by radioreceptorassay in liver membranes. The peaks observed with this method in samples from human bowel have also been analysed in two other assays: stimulation of cyclic AMP accumulation in gastric glands and radioimmunoassay. Owing to the different patterns of activity of porcine G-37 and G-29 in these assays, the comparison of the data obtained allows to discriminate between the two peptides. The same behaviour in both HPLC and the three assays of the human peaks on one hand and the porcine peptides on the other strongly suggests that human intestine contains a very similar or the same molecules as that isolated from the porcine tissues. Whatever the portion of small intestine, G-37 represented ca 90% of G-37 + G-29. A decreasing concentration gradient of both G-37 and G-29 was also observed from ileum to descending colon.

Increased gastric acid secretion occurs after extensive intestinal resection in man, dog, rat, and monkey. Hypergastrinemia has been observed in patients with short gut syndrome and appears to accompany the hyperacidity after intestinal resection in dog, rat, and monkey. Postresectional hypergastrinemia is caused by increased release of gastrin and/or decreased degradation of the hormone. Other hormonal changes after extensive resection include increased insulin, GIP, pancreatic glucagon, and decreased enteroglucagon.