Synthesis and secretion of glucagon-like peptide-1 by fetal rat intestinal cells in culture

Secretion of the intestinal proglucagon-derived peptides (PGDPs) including the incretin glucagon-like peptide-1 (GLP-1) is regulated, at least in part, by the duodenal hormone glucose-dependent insulinotropic peptide (GIP) through a protein kinase (PK) A-dependent pathway. It has been demonstrated that the activation of PKA increases the synthesis of some intestinal PGDPs, particularly the glucagon-like immunoreactive (GLI) peptides glicentin and oxyntomodulin. However, the effects of GIP on GLI and GLP-1 synthesis are not known. Fetal rat intestinal cells in culture were therefore treated for up to 24 h with 5mm dbcAMP or 10⁻⁶ m GIP and the changes in glicentin, oxyntomodulin, GLP-1^x-37 and GLP-1^x-36NH2 secretion and synthesis were examined by RIA and HPLC. Both dbcAMP and GIP increased the acute (2 h; to 224±21 and 256±20% of controls, respectively,P<0.001) and chronic (24 h; to 230±22 and 130±6% of controls, respectively,P<0.001) secretion of intestinal PGDPs. In contrast, the total culture content of PGDPs was increased only after 24 h of incubation (to 156±15 and 125±7% of controls for dbcAMP and GIP, respectively,P<0.01). HPLC analysis confirmed that the intestinal cultures produced the GLI peptides glicentin and oxyntomodulin, as well as the biologically active forms of GLP-1, GLP-7^7–37 and GLP-1^7-36NH2. The relative proportion of these peptides was not altered by treatment with dbcAMP or GIP. Thus, in addition to its effects on GLP-1 release from the rat intestine, GIP appears to be an important regulator of the synthesis of this insulinotropic peptide.     

Comparative study in vivo and in vitro of the differentiation of immunoreactive corticotropic cells in fetal rat anterior pituitary

In order to study the mechanisms of the differentiation of adenohypophysial corticotropic cells, an immuno-cytological study was performed in fetal rat anterior pituitary in vivo and in vitro with antisera against beta-(1-24) and alpha-(17-39) ACTH and beta-LPH, alpha- and beta-endorphins. In vivo, these cells appeared at 16 days of gestation without any difference in the timing of appearance of the two immunoreactivities. The same immunoreactivity was also detected in adenohypophysial primordia explanted from 12 to 15 days of gestation and maintained in organ culture until 21 days by using either medium containing fetal calf serum or medium containing insulin and transferrin instead of fetal calf serum. These immunoreactive cells were first detected in the different experimental primordia after a minimal period of culture, corresponding to a final equivalent of 16 days as in vivo. However, the mean cytoplasmic area of immunoreactive cells increased in relation to the day of explantation whatever the duration of culture. These data suggest: (1) the nature of culture medium used in this study has no influence on the differentiation of the corticotropic cells; (2) this cell type seems to be committed precociously (before day 12) by one or several substances of unknown origin; (3) the normal development seems to require the presence of factors (before day 14) whose nature and origin remain to be elucidated.     

Human intestinal goblet cells in monolayer culture: characterization of a mucus-secreting subclone derived from the HT29 colon adenocarcinoma cell line

HT29-18N2 (N2) cells, a subclone of the HT29 human colon carcinoma cell line, are shown in this report to be a model system for the study of human goblet cell differentiation and mucin secretion. Grown in the absence of glucose, these cells formed homogeneous epithelial monolayers of columnar cells with typical goblet cell morphology. Differentiation occurred on uncoated glass; laminin, fibronectin, or collagen type I or IV did not enhance differentiation. HT29-18N2 cells grown on uncoated or matrix-coated permeable filters formed differentiated monolayers, but mucin granules within some of these cells polarized along intraepithelial lumens. Polyclonal antibodies raised against purified human colonic mucin, and also a monoclonal antibody against a protease-sensitive epitope of human colonic mucin, stained secretory granules of all differentiated goblet cells within N2 cell monolayers but did not stain predifferentiated goblet cells lacking large secretory granules. Monoclonal antibodies against specific carbohydrate sequences of human mucins also failed to stain N2 cells before differentiation, but recognized varying fractions of differentiated N2 goblet cells. Autoradiographic visualization of radiolabeled glycoproteins demonstrated transport and secretion of N2 cell mucin granules. Cholinergic stimulation of differentiated N2 cell monolayers resulted in depletion of intracellular mucin granules.     

Fetal rat chondrocytes sequentially elaborate separate growth- and differentiation-promoting peptides during their development in vitro

Chondrocytes isolated from the calvaria of rat fetuses proliferate and form cartilage when cultured in a chemically defined, serum-free medium, suggesting that they may elaborate self-regulatory factors. Conditioned media obtained from these chondrocytes stimulated DNA synthesis and proliferation when added to separate cultures of chondrocytes and the closely related osteoblast-like cells, but were not very effective in skin fibroblasts isolated from the same fetuses, as judged by [3H]thymidine incorporation and cell proliferation. Chondrocyte-conditioned medium also promoted chondrocyte differentiation, augmenting 35SO4 incorporation, and the accumulation of type II collagen and cartilage-specific proteoglycan. Stimulation of growth and differentiation appears to be attributable to separate activities, released into the medium sequentially by the cultured chondrocytes during their proliferation and maturation phases. Media obtained from growing chondrocytes stimulated growth, but had little effect on 35SO4 incorporation. Media obtained from mature cultures promoted growth as well as 35SO4 incorporation. The mitogenic and sulfation activities were trypsin inhibitable, but exhibited different solubility characteristics and striking differences in their patterns of elution from gel filtration columns. These results suggest that chondrocytes elaborate autostimulatory peptides, the biological activities of which mirror, at least in part, the developmental stage of the donor cells.     

Organization of angiotensin II immunoreactive cells and fibers in the rat central nervous system. An immunohistochemical study

The distribution of angiotensin II (AII)-immunoreactive cells and fibers was examined in adult male Sprague-Dawley rats with and without colchicine pretreatment. As seems to be the case for a number of other neuropeptides, AII is preferentially found in brain stem, hypothalamic, and limbic structures involved in the control of homeostatic functions. AII-stained cell bodies were most prominent in magnocellular parts of the paraventricular and supraoptic nuclei, and cells were also found in parvocellular parts of the former. Other hypothalamic nuclei containing cell bodies include the suprachiasmatic nucleus, the medial preoptic area, and perifornical parts of the lateral hypothalamic area. Of considerable interest was robust staining in several of the circumventricular organs, in particular the subfornical organ, where both cells and fibers were found. The results of water deprivation and nephrectomy suggest that this staining does not represent uptake of circulating peptide, but instead, represents AII-containing neural connections. In the thalamus, AII-stained cells were found in the paraventricular nucleus, the central medial nucleus, the nucleus reuniens, and rostral parts of the zona incerta. Two cell groups in the basal telencephalon, in the dorsal part of the bed nucleus of the stria terminalis and in the medial nucleus of the amygdala, lay at either end of an AII-stained pathway coursing through the stria terminalis. In the midbrain, immunoreactive cells were found in the interpeduncular and peripeduncular nuclei, and one pontine cell group was detected in the most lateral part of the lateral parabrachial nucleus. The only AII-stained cells in the medulla were in the nucleus of the solitary tract, near the margin of the area postrema. Fibers were found at all levels of the central nervous system, from the olfactory bulbs to the spinal cord, where terminal fields were observed in the substantia gelatinosa and in the intermediolateral cell column. Longitudinally oriented fibers were present throughout the periventricular fiber system and in the medial forebrain bundle, including its caudal extension in ventrolateral parts of the brain stem. It is suggested that, at many different levels, AII serves as both a hormone and neurotransmitter for fluid balance.     

Role of the adrenal renin-angiotensin system on adrenocorticotropic hormone- and potassium-stimulated aldosterone production by rat adrenal glomerulosa cells in monolayer culture

The rat zona glomerulosa has a renin-angiotensin system that appears to function as an autocrine or paracrine system in the regulation of aldosterone production. To further investigate dynamic changes of production of renin and aldosterone in vitro we developed a primary monolayer culture of rat adrenal glomerulosa cells in serum-free medium. Collagenase-dispersed glomerulosa cells were incubated in PFMR-4 medium containing 10% fetal calf serum for 48 hours; the medium was then replaced with serum-free PFMR-4 medium. The cell viability and the aldosterone secretion were stable over the additional 48 hours in the serum-free control medium. After incubation for 24 hours in the serum-free medium, the cells were exposed to high K+ or adrenocorticotropic hormone (ACTH) for another 24 hours. ACTH stimulated aldosterone secretion, and this increased secretion was associated with an increase in renin activity (cell active renin, from 15.56 +/- 0.71 to 45.75 +/- 5.69; cell inactive renin, from 0.67 +/- 0.54 to 8.75 +/- 3.40; medium inactive renin, from 5.58 +/- 1.16 to 106.20 +/- 14.01 pg angiotensin I (Ang I)/micrograms protein/3 hr). Aldosterone was also stimulated by high K+. This increase was also associated with an increase in active renin in the cells (from 15.08 +/- 1.80 to 23.26 +/- 2.15 pg Ang I/micrograms protein/3 hr) and an increase in inactive renin in the medium (from 10.87 +/- 1.62 to 21.37 +/- 3.20 pg Ang I/micrograms protein/3 hr). Addition of the angiotensin converting enzyme inhibitor lisinopril attenuated both ACTH- and high K(+)-stimulated aldosterone secretion significantly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth in monolayer culture of rat pituitary cells which synthetize and release acth.

Cultures of rat pituitary gland cells were developed to study biosynthesis and release of ACTH. ACTH measurement was accomplished by radioimmunoassay. ACTH release was observed following stimulation with theophylline and cAMP in a dose-dependent manner. Biosynthesis was demonstrated by incorporation of 3-H-phenylalanine into the hormone, employing a double antibody technique.     

In vitro study of Starling's hypothesis in a cultured monolayer of bovine aortic endothelial cells

Starling's hypothesis that fluid movement across the microvascular wall is determined by the transmural differences in hydrostatic and osmotic pressures was tested using an in vitro model comprised of bovine aortic endothelial cells grown on a porous support. In all experiments, a 1% bovine serum albumin (BSA) solution was maintained in the abluminal reservoir and the luminal reservoir contained either a 1 or a 5.5% BSA solution. The global osmotic pressure difference across the endothelial layers was thus either 0 or 20.3 cm H2O. When the luminal concentration of BSA was changed from 1 to 5.5% at a hydrostatic pressure differential of 5, 10 or 20 cm H2O, no reverse flow (in the reabsorption direction) was observed even though the hydrostatic pressure differential was far below the global osmotic pressure differential. In another case, the hydrostatic pressure differential was dropped quickly from 20 to 5 cm H2O, while a constant osmotic pressure differential was maintained by 5.5% BSA in the luminal reservoir. A strong transient reabsorption flow was observed over a 30-second period which diminished to undetectable levels within 2.5 min; then a sustained steady-state filtration flow was observed after 20 min. These in vitro experiments support other studies in capillaries showing transient reabsorption that decays to steady-state filtration at longer times.     

Glucagon-like immunoreactive peptides in a rat ileal epithelial cell line (IEC-18)

The presence of cells containing glucagon-like immunoreactive (GLI) peptides was demonstrated in a rat ileal epithelial cell line (IEC-18) by both immunofluorescence and radioimmunoassay. When cell extracts were subjected to gel filtration chromatography, the cells were found to contain 3.5 Kd glucagon in addition to significant quantities of large molecular weight GLI peptides (apparent molecular weights of 4, 6, 8 and 10 Kd) and a 9 Kd peptide with apparent glucagon immunoreactivity. This was in contrast to extracts of adult rat ileum, which contained only large molecular weight GLI peptides (apparent molecular weights of 6 and 12 Kd). Production of GLI peptides by the IEC-18 cells was stimulated by glucose (p less than 0.02) and inhibited by insulin (p less than 0.01). In conclusion, these results demonstrate that the IEC-18 cells produce both GLI peptides and glucagon, and thus support the notion that proglucagon processing is cell-specific. IEC-18 cells may therefore provide a tool for investigations of some aspects of GLI peptide and glucagon synthesis.     

Organization of ovine corticotropin-releasing factor immunoreactive cells and fibers in the rat brain: an immunohistochemical study

The distribution of corticotropin-releasing factor (CRF)-immunoreactive cells and fibers has been examined in the brains of normal adult rats, and in the brains of animals that had been pretreated with intraventricular injections of colchicine, or had been adrenalectomized 3-60 days before perfusion. The results suggest that CRF immunoreactivity is localized in at least three functionally distinct systems. First, most of the CRF-stained fibers in the neurohemal zone of the median eminence, which presumably modulate the release of ACTH and beta-endorphin from the pituitary, appear to arise in the paraventricular nucleus of the hypothalamus (PVH). About 2,000 CRF-stained cells are distributed throughout all eight parts of the PVH, although a majority (80%) of the cells are concentrated in the parvocellular division, and a smaller number (about 15%) are found in parts of the magnocellular division in which oxytocinergic cells predominate. This appears to be the only CRF-stained pathway in the brain that is affected (increased staining intensity) by adrenalectomy. Second, a series of cell groups in the basal telencephalon, hypothalamus, and brain stem that are known to play a role in the mediation of autonomic responses contain CRF-stained neurons. These areas, which are interconnected by stained fibers in the medial forebrain bundle and the periventricular system, include the central nucleus of the amygdala, substantia innominata, bed nucleus of the stria terminalis, medial and lateral preoptic areas, lateral hypothalamic area, central gray, laterodorsal tegmental nucleus, locus ceruleus, parabrachial nucleus, dorsal vagal complex, and regions containing the A1 and A5 catecholamine cell groups. And third, scattered CRF-stained cells are found throughout most areas of the cerebral cortex. Most such cells are confined to layers II and III in the neocortex, and their bipolar shape suggests that they are interneurons. These cells are most common in limbic regions including prefrontal areas, the cingulate gyrus, and areas bordering the rhinal fissure. Scattered immunoreactive cells are also found in dorsal parts of the dentate gyrus and Ammon's horn. These results suggest that the PVH plays a critical role in the modulation of ACTH and beta-endorphin release from the pituitary, and that CRF-containing pathways in the brain are involved in the mediation of autonomic responses.     

Usefulness of the organ culture system in the in vitro diagnosis of coeliac disease: a multicentre study

OBJECTIVE: Diagnosis of coeliac disease is based on the presence of villous atrophy which recovers following a gluten-free diet. The presence of circulating antiendomysial antibodies as well as their disappearance after a gluten-free diet supports the diagnosis. It has also been demonstrated that antiendomysial antibodies are detectable in supernatants of cultured intestinal biopsies from patients with coeliac disease. The objective of this study was to compare the histology and antiendomysial antibodies in culture supernatants of intestinal biopsies to validate the in vitro organ culture system as a future diagnostic tool for coeliac disease. MATERIAL AND METHODS: Seventy-five antiendomysial serum-positive patients on a gluten-containing diet were evaluated. Patients underwent endoscopy with 5 biopsy fragments: 3 for histology, 1 cultured with and the other without gliadin-peptide activator. Antiendomysial antibodies were evaluated in all culture supernatants. RESULTS: Sixty-eight patients had evidence of villous atrophy, while 73 out of 75 were positive to the organ culture system. The agreement rate between organ culture and histology results was 94%. CONCLUSIONS: As all the centres participating in the study obtained good agreement between organ culture and histology results, the new system could be considered a reliable tool for the diagnosis of coeliac disease. Nevertheless, it is possible to highlight cases with an organ culture-positive and -negative histology. This feature could be of considerable interest because, as the sensitivity of organ culture seems to be greater than the initial histology, the new system might be useful in uncertain cases where the risk of missing the diagnosis of coeliac disease is high.     

Synthetic alpha-subunit peptides stimulate testosterone production in vitro by rat Leydig cells.

The glycoprotein hormones (LH, hCG, FSH, and TSH) have a common 92-amino acid alpha-subunit which is noncovalently linked to a hormone-specific beta-subunit. Synthetic peptides of the alpha-subunit have been shown to inhibit binding of [125I]iodo-hCG to rat ovarian membrane and [125I]iodo-TSH to human thyroid membrane preparations. Synthetic overlapping peptides of the alpha-subunit of hCG were prepared by solid phase techniques and tested in a standard in vitro rat Leydig cell bioassay. Three regions in the alpha-subunit (alpha 1-15, alpha 30-45, and alpha 71-85) were found to stimulate testosterone production. All three regions correlate with inhibition of hCG binding to ovarian receptors, but subtle differences exist between the binding sites and effector sites. These data indicate that the glycoprotein alpha-subunit has intrinsic bioactivity.     

Neonatal T cells as a model system to study the possible in vitro senescence of lymphocytes

In this study long-term neonatal T-lymphocyte cultures were initiated from cord blood following alloantigenic stimulation. Growth curves and population doublings were measured for replicate cultures, functional and phenotypic analyses performed, and cells were cloned. Thus, newborn T cells were shown to constitute a potentially excellent model for the analysis of possible in vitro senescence of immunologically relevant cells. Certain problems of the system centering around "crisis" periods and reproducibility, were additionally explored.     

Diabetic intestinal growth adaptation and glucagon-like peptide 2 in the rat: effects of dietary fibre

BACKGROUND/AIMS: Dietary fibre influence growth and function of the upper gastrointestinal tract. This study investigates the importance of dietary fibre in intestinal growth in experimental diabetes, and correlates intestinal growth with plasma levels of the intestinotrophic factor, glucagon-like peptide 2 (GLP-2). METHODS: Male Wistar rats were randomised to the following groups: two streptozotocin-diabetic and two control groups fed either a fibre-containing or a fibre-free diet for three weeks. Intestinal weight, length, and morphometric data (villus height, villus area, crypt depth) were measured. Blood samples were obtained after two weeks for measurement of GLP-2 and enteroglucagon (glicentin, oxyntomodulin). RESULTS: The mean daily consumption of food in the two diabetic groups was 40% higher than in controls. In diabetic rats fed fibre, the increase in intestinal weight from day 0 to 20 was sixfold greater than that of the controls and small intestine weight per cm length was increased by 50%. In the diabetic rats fed a fibre-free diet, intestinal growth was 30% less than in diabetic rats fed fibre, and intestinal weight increased only threefold compared with controls. Morphometric data showed that the intestinal increase in diabetic rats fed fibre was due primarily to growth of the mucosal layer. Villus height and crypt depth increased 60% and 40% respectively, but by only 20% in fibre-free diabetic rats. The plasma levels of GLP-2 parallelled diabetic intestinal growth, whereas plasma levels of enteroglucagon increased regardless of the extent of intestinal growth. CONCLUSIONS: Intestinal growth in experimental diabetes is strongly influenced by the presence of dietary fibre. The effect may be mediated by GLP-2.