Effect of somatostatin on small intestine enzyme activities in rat and chick

The influence of somatostatin injection on intestinal disaccharidase and alkaline phosphatase activity in rat and chick was investigated. Disaccharidase and alkaline phosphatase activities of rat and chick homogenates were not modified. In rat the activities of mucosal brush-border maltase and sucrase were significantly increased. In chick brush-border a significant increase of duodenal mucosal activity and duodenal and jejunal sucrase activity is observed.     

Intestinal brush border membrane-bound disaccharidases of the American alligator, Alligator mississippiensis

1. The disaccharidase activities of the small intestines of American alligators (Alligator mississippiensis) were studied in epithelial scrapes and brush-border membrane preparations. 2. Maltase, isomaltase and trehalase activities were found. Activities of these enzymes were higher in the proximal small intestine and decreased distally. 3. Disaccharidase activities were enriched 12-15 times in brush-border membrane preparations, compared with mucosa/enterocyte crude homogenates and were co-enriched with the brush-border membrane marker alkaline phosphatase. 3. The pH optima were: maltase 6.5; isomaltase 5.6; and trehalase 5.8. The Q10 of maltase, the most active enzyme, was equal to 1.82. 4. In reptiles, as in mammals, disaccharidase activities may be correlated with feeding habits. The co-occurrence of sucrase and isomaltase may not be a common feature of vertebrates.     

Alkaline phosphatase and peptidase activities in Caco-2 cells: Differential response to triiodothyronine

Summary Caco-2 cell human colon adenocarcinoma cell line was used to study the hormonal regulation of small intestinal epithelial cell differentiation. We had previously shown that insulin-transferrin-selenium and triiodothyronine (5 × 10⁻⁸ M)-supplemented medium can best replace serum after 2 days of culture for both the maintenance and differentiation of Caco-2 cells. The present study demonstrates that precoating petri dishes with complete serum allows the growth and differentiation of Caco-2 cells seeded directly in serum-free medium. On the other hand, precoating with dialyzed serum inhibits alkaline phosphatase and dipeptidyl-dipeptidase IV activities by more than 50%. The results obtained with complete serum-precoated culture plates indicate that there is no synergy between insulin and triiodothyronine because cells maintained in transferrin-selenium and triiodothyronine-supplemented medium, with or without insulin, express comparable enzyme activities. Moreover, large increases in alkaline phosphatase and dipeptidyl-dipeptidase IV activities were observed when triiodothyronine was added to the culture medium by the time confluency was reached. In contrast, γ-glutamyltransferase was lowered to a greater extent when triiodothyronine was present from the beginning of culture. These findings show that triiodothyronine preferentially stimulates alkaline phosphatase and dipeptidyl-dipeptidase IV activities during the differentiation period whereas it selectively inhibits γ-glutamyltransferase during the proliferation phase. Triiodothyronine acts in a dose-dependent manner.     

Effects of lactoferrin on intestinal epithelial cell growth and differentiation: an in vivo and in vitro study

This study was designed to analyse the effects of human (h) and bovine lactoferrin (bLF) on the growth and differentiation of intestinal cells using the mice model supplemented with Lactoferrin (LF) and the enterocyte-like model of Caco-2 cells which spontaneously differentiate after confluency. In mice, bLF supplementation increased jejunal villus height and the expression of several intestinal brush border membrane enzymes activities. Addition of bLF or hLF to undifferentiated Caco-2 cells was able to increase cell proliferation with confluency being reached more rapidly. Moreover, when Caco-2 cells were grown in the presence of LF for 3 weeks, brush-border membrane-associated enzyme activities i.e. sucrase, alkaline phosphatase and neutral aminopeptidase, as well as the l-glutamate transporter expression were all increased indicating an increased Caco-2 cell differentiation. Accordingly, cDNA Atlas array and Western blot analysis of cell cycle proteins shown a decreased expression of Cdck2 and an increased TAF1 expression; these proteins being implicated in the regulation of numerous genes related to cellular proliferation and differentiation. These modifications were associated with an inhibition of Caco-2 cell spontaneous apoptosis. Altogether, our results indicate that LF increase in vivo and in vitro enterocyte differentiation. In addition, LF was found to increase in vitro enterocyte proliferation resulting in higher cell density in cell flasks, an effect that was likely partly due to a reduction of the cellular apoptosis. The different stimulation patterns observed for the different parameters associated with cell differentiation in relationship with specific gene regulation is discussed.     

Enterocytic differentiation and glucose utilization in the human colon tumor cell line Caco-2: modulation by forskolin

The human colon cancer line Caco-2 exhibits after confluency a concomitant increase of glycogen accumulation and an enterocytic differentiation. The purpose of this work was to investigate whether forskolin (FK), an activator of adenylate cyclase, would induce a permanent glycogenolysis and, if so, whether it would result in modifications of the differentiation pattern of the cells. FK activates adenylate cyclase in Caco-2 cells with an ED50 of 7 X 10(-6)M. Three different treatment protocols with FK (10(-5)M) were applied: 1) the cells were treated during all the time in culture (20 days); 2) the treatment was started after confluency; 3) the treatment was interrupted after confluency. The presence of FK results in a permanent stimulation of cAMP accumulation (10 to 20 fold the basal values) and in a permanently reduced glycogen content (30 or 50% of the control values). The rates of glucose consumption are increased three and five fold in protocols 1 and 3 respectively. These metabolic changes are associated with morphological changes (tightening of the intercellular spaces and shortening of the brush border microvilli) and with a dual inhibition of the activities of brush border hydrolases: a) an inhibition of the post-confluent increase of activity of sucrase, aminopeptidase N and alkaline phosphatase in the brush border enriched fraction; b) an inhibition of the post-confluent increase of activity of sucrase in the cell homogenate. A comparison of the results obtained in each protocol shows that the morphological modifications and the decrease of the enzyme activities in the brush border fraction are regularly associated with an increased cAMP accumulation, whereas the inhibition of the differentiation of sucrase is a direct consequence of the increase in glucose consumption and decrease in glycogen stores.     

Comparative study of the effect of hydrocortisone and thyroxine on suckling mouse small intestine in organ culture

The influence of hydrocortisone (10(-8)--10(-5) M) and thyroxine (10 (-9)--10(-6) M) on intestinal epithelial cell differentiation and proliferation have been studied using explants of suckling mouse jejunum maintained in serum-free organ culture. Hydrocortisone induced the appearance of sucrase activity and increased trehalase, glucoamylase, lactase and alkaline phosphatase activities. Thyroxine was completely ineffective at all the concentrations used. None of these hormones affected the mitotic activity or the 3H-thymidine incorporation into DNA. These results demonstrate that hydrocortisone but not thyroxine acts directly on intestinal brush border membrane differentiation and that both hormones do not influence the proliferation of the epithelial cells during postnatal development.     

Chromatofocussing and centrifugal reconstitution as tools for the separation and characterization of the Na+-cotransport systems of the brush-border membrane

Chromatofocussing was used for the separation of brush-border membrane proteins from calf kidney into 4 or 5 fractions over the pH range 4.0 to 7.4. These fractions were reconstituted into proteoliposomes by gradient centrifugation. Determination of the sodium-dependent solute uptake by proteoliposomes reconstituted from different chromatofocussed fractions showed that the sodium-D-glucose cotransport system was present in the fraction eluted between pH 5.3 and 5.8, and that the sodium-phosphate cotransport was present in fractions eluted between pH 4.6 and 5.3 and between pH 5.8 and 6.6, sodium-alanine cotransport could be detected in almost all fractions. Marker enzymes of the brush-border membrane, such as alkaline phosphatase, gamma-glutamyltransferase and aminopeptidase M etc. were also found to be eluted at pH 7.0-7.4, 4.0-4.1 and 5.6-5.8, respectively. These results suggest that chromatofocussing is a promising tool for the separation of membrane proteins and for pre-purification of the sodium-D-glucose cotransport system. It can be further concluded that the sodium-dependent phosphate transport across the brush-border membrane is not dependent upon alkaline phosphatase activity.     

Bifidobacterium bifidum monoassociation of gnotobiotic mice: Effect on enterocyte brush-border enzymes

The effect of intestinal colonization withBifidobacterium bifidum (Gram-positive anaerobic bacterium colonizing the intestine of healthy new-born mammals, exhibiting a probiotic effect, protecting the intestinal mucosa against colonization by pathogenic microflora) on enterocyte brush-border enzymes was examined in weaned 23-d- and in 2-month-old gnotobiotic inbred mice and compared with that in corresponding germ-free (GF) and conventional (CV) controls. The two groups of GF mice were associated with humanB. bifidum 11 d before the end of the experiment. Specific activity of enterocyte brush-border enzymes—lactase, alkaline phosphatase and γ-glutamyltranspeptidase was significantly higher in both age groups of GF mice in comparison with CV ones; on the other hand, sucrase and glucoamylase activities were higher in CV mice. Monoassociation withB. bifidum accelerates biochemical maturation of enterocytes resulting in a shift of specific activities of brush-border enzymes between the values found for GF and CV mice. This effect ofB. bifidum supplementation was less pronounced for alkaline phosphatase, sucrase, glucoamylase and dipeptidyl peptidase IV in immature gut of weaned mice than of 2-month-old ones.     

Activity of renal hydrolases in pre- and postnatal development of mice

The fetal and postnatal activity patterns of different hydrolytic enzymes (alkaline phosphatase, gamma-glutamyltransferase, trehalase, maltase, glucoamylase, lactase, and sucrase) have been examined in mouse renal homogenates. Alkaline phosphatase and gamma-glutamyltransferase activities presented approximately similar changes. They increased from 18 days of gestation up to 30 days after birth. These activities showed marked increases during the 3rd and 4th postnatal weeks. A similar important rise was observed for trehalase activity at the end of the suckling period. Maltase activity increased gradually after birth. Traces of lactase, sucrase, and glucoamylase activities were detected at each developmental stage.     

Effect of pentagastrin, secretin and cholecystokinin on growth and differentiation in organ cultured rabbit small intestine

The effect of pentagastrin, secretin and cholecystokinin on biochemical parameters of mucosal growth and differentiation was studied in organ cultured rabbit jejunum and ileum. Pentagastrin at 0.05-5.0 microgram/ml did not affect DNA content of the biopsy, but led to a significant decrease of sucrase and alkaline phosphatase activity in the ileum. Secretin prompted a significant decrease of DNA and protein in the ileum at a level of 10(-7) and 10(-5) M, but had no effect in the jejunum. Of the brush border enzymes, sucrase and alkaline phosphatase were suppressed in both parts of the intestine both with respect to specific activity and total biopsy content. Cholecystokinin, like pentagastrin, did not influence DNA or protein content, but reduced sucrase, maltase and alkaline phosphatase activity. HMG-CoA reductase, the key enzyme of cholesterol synthesis, was not significantly affected by any of the three hormones tested. When brush border enzymes or DNA from desquamated cells were measured in the post-culture medium, no consistent effect of any gastrointestinal hormone was apparent. The present study demonstrates a direct "antitrophic" effect of secretin in cultured mucosa. Pentagastrin and cholecystokinin did not influence mucosal DNA content in vitro but apparently inhibited villus cell differentiation.     

Differential effect of Bacillus firmus on immune response and enterocyte brush-border enzyme levels in BALB/c and B10.BR mice

A nonpathogenic bacterium of external environment possessing remarkable immunomodulatory activity, Bacillus firmus (BF) inactivated with formaldehyde, was given intragastrically to two genetically different mouse strains BALB/c (H-2d) and B10.BR/SnPh (B10.BR, H-2k) reared in conventional (CV) and B10.BR strain also in germ-free (GF) conditions. Repeated intragastric administration of BF (500 micrograms every other day over two weeks, starting at the age of 3 months) significantly enhanced intestinal IgA levels in CV BALB/c mice but did not affect intestinal IgA in CV B10.BR mice. In GF B10.BR mice, IgG levels in sera and intestinal washings increased after BF administration compared to CV B10.BR mice. In CV BALB/c mice, specific activity of enterocyte brush-border enzymes (lactase, gamma-glutamyltransferase, alkaline phosphatase) decreased after BF treatment; sucrase (sucrose alpha-glucosidase) activity was not affected. On the other hand, in B10.BR mice, specific activity of gamma-glutamyltransferase and dipeptidyl peptidase IV were higher after administration of BF in both CV and GF groups relative to untreated controls. The activities of lactase and glucoamylase (glucan 1,4-alpha-glucosidase) were significantly stimulated only in the group of GF B10.BR mice treated with formolized BF. The stimulation of immunoglobulin production after BF treatment was accompanied by changes in the levels of enterocyte brush-border enzymes; this responsiveness to BF treatment was genetically regulated.     

Organ culture of suckling rat intestine: comparative study of various hormones on brush border enzymes

Jejunal mucosa of 6 d-old rats were cultured for 24 and 48 h in the presence of thyroxine, insulin, pentagastrin, glucagon, epidermal growth factor (EGF) or dibutyryl-A-3:5-MP cyclic with or without dexamethasone (DX). The enzymes were assayed on the purified brush borders. The various agents added alone to the basic culture medium had no effect with the exception of DX on the levels of enzyme activities. Dexamethasone alone induced sucrase, stimulated maltase, and protected other brush border enzyme activities (aminopeptidase, lactase, and alkaline phosphatase). When added to DX-supplemented medium, only the following factors modified the levels of enzymatic activities observed with DX alone. Insulin (10(-6) M) increased maltase, alkaline phosphatase, and lactase activity to a greater extent than DX at 24 h culture, the effect being maintained at 48 h on alkaline phosphatase only. At 48 h culture, both EGF (10(-8) M) and dbcAMP (10(-3) M) decreased DX-induced sucrase activity. The latter agent also depressed DX-stimulated aminopeptidase activity.     

Sodium-dependent D-glucose transport in brush-border membrane vesicles from isolated rat small intestinal villus and crypt epithelial cells

Differentiation and maturation of enterocytes occur with migration from the crypt to villus compartments. To investigate the effect of epithelial cell differentiation on sodium-dependent D-glucose transport, brush-border membrane vesicles were prepared from small intestinal epithelial cell suspensions selectively isolated from villus and crypt populations. Enterocytes were isolated with a morphologically monitored sequential cell dissociation method. Thymidine kinase, sucrase, and alkaline phosphatase activities were measured as differentiation markers of specific cell populations. Brush-border membrane vesicles were purified and their kinetic characteristics defined with a rapid filtration method under conditions of a zero-trans, 100 mM cis-NaSCN gradient. Typical "overshoot" phenomena characteristic of sodium D-glucose cotransport were observed for both villus (five- to eight-fold equilibrium values) and crypt brush-border membrane vesicles (two- to four-fold equilibrium values). Kinetics analyses of the initial D-glucose flux in brush-border membrane vesicles suggested the presence of at least two sodium-dependent D-glucose carriers in the villus and only a single carrier in the crypt compartments. These data indicate that sodium D-glucose cotransport occurs in brush-border membranes of both villus and crypt populations. Moreover, quantitative and qualitative differences between these two membrane populations suggest that epithelial D-glucose transport processes are differentiation dependent and reflect the degree of enterocyte development.     

Characterization of brush borders purified in iso-osmotic medium and microvillar membranes subfractionated from mouse small intestine.

Brush borders free of nuclei were isolated by repeated homogenization and centrifugation in iso-osmotic medium. They showed typical morphology under electron microscopy. The mean recovery and enrichment of alkaline phosphatase activity in the brush-border fraction were 50% and 17.5-fold respectively. gamma-Glutamyl transpeptidase showed a close parallelism with alkaline phosphatase and sucrase in subcellular distribution. Microvillar membranes were purified from isolated brush borders; they showed a further enrichment for alkaline phosphatase and were composed of homogeneous vesicles. Both brush-border and microvillar-membrane preparations were analysed for contamination by basolateral and endoplasmic-reticular membranes. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the microvillar-membrane preparation in six different systems revealed approx. 40 components in the mol.wt. range 15 000-232 000. They were grouped into seven major classes on the basis of molecular weight and electrophoretic patterns.     

Expression of the H type 1 blood group antigen during enterocytic differentiation of Caco-2 cells.

We made a comparative study of the structures of the oligosaccharides on the glycoproteins from Caco-2 human colonic adenocarcinoma cells, before and after differentiation. Enterocytic differentiated Caco-2 cells highly express H type 1 blood group antigen on the cell surface as well as activities of brush border membrane hydrolases, such as dipeptidyl peptidase IV and alkaline phosphatase. A strong correlation was observed between the amounts of H type 1 blood group antigen and the degrees of differentiation. Structural analysis with use of lectin affinity high performance liquid chromatography revealed that typical mucin-type sugar chains of the glycoproteins from undifferentiated cells have H type 2 group, linear polylactosamines, and core 1 structure. On the other hand, differentiated cells newly contain H type 1 and Le(b) groups and core 2 structure. Mucins with H type 1 group make contact with brush border membrane enzymes on differentiated cells. Furthermore benzyl 2-acetamide-2-deoxy-alpha-D-galactopyranoside inhibited both expression of H type 1 group on the cell surface and enhancement of brush border membrane enzyme activities even in the presence of a differentiating inducer. These results suggest that the mucin-type sugar chains with H type 1 group have important functions regarding differentiation of Caco-2 cells.     

Acute dieldrin toxicity: effect on the uptake of glucose and leucine and on brush border enzymes in monkey intestine

Administration of a single oral dose of dieldrin (20 mg/kg body wt.) to rhesus monkeys considerably elevated the uptake of glucose and the activities of brush border sucrase, lactase, maltase and alkaline phosphatase in intestine compared to control animals. Leucine uptake and leucine amino peptidase activity was significantly depressed in pesticide-treated animals. Kinetic studies with brush border sucrase revealed that augmentation of enzyme activity in pesticide-fed animals was due to an increase in the disaccharidase content.     

Influences of dexamethasone on the maturation of fetal mouse intestinal mucosa in organ culture

The effects of glucocorticoids on the maturation of the fetal small intestinal mucosa have been studied using duodenal explants resected at 17 days of gestation and cultured in a serum-free medium in the presence or absence of dexamethasone (30-300 ng/ml). Dexamethasone (a) increases specifically alkaline phosphatase, maltase, trehalase and sucrase activities and (b) allows an accumulation of goblet cells along the villi at a faster rate than that occurring in utero. These results indicate that glucocorticoids influence directly the differentiation of absorptive cells and goblet cells in the small intestine during the fetal period.     

Protective effects of nocardia delipidated cell mitogen on the mucosa of the small intestine after irradiation of germ-free piglets.

The radioprotective effect of the bacterial immunomodulator Nocardia delipidated cell mitogen (NDCM) on intestinal mucosa and disaccharidase activities was studied in irradiated germ-free piglets. Three-week-old germ-free (GF) piglets were intragastrically pretreated with 1 mg NDCM per 1 kg body weight. The piglets were whole-body irradiated with 2.5 Gray five days after the NDCM pretreatment and sacrificed eight days after irradiation. In the non-irradiated group of GF piglets, NDCM application stimulated lactase activity and markedly increased sucrase activity. This stimulatory effect of NDCM disappeared after irradiation and the piglets exhibited a normal activity of lactase in the jejunal brush-border membrane vesicles, while the sucrase activity decreased to the level found in irradiated controls. NDCM-pretreated intestinal mucosa contained some infrequent lymphocytes which disappeared from the control irradiated tissue. It also exhibited less injury of the epithelium and stroma cells.     

Regulation of Human (Caco-2) Intestinal Epithelial Cell Differentiation by Extracellular Matrix Proteins

Extracellular matrix regulation of intestinal epithelial differentiation may affect development, differentiation during migration to villus tips, healing, inflammatory bowel disease, and malignant transformation. Cell culture studies of intestinal epithelial biology may also depend on the matrix substrate used. We evaluated matrix effects on differentiation and proliferation in human intestinal Caco-2 epithelial cells, a model for intestinal epithelial differentiation. Proliferation, brush border enzyme specific activity, and spreading were compared in cells cultured on tissue culture plastic with interstitial collagen I and the basement membrane constituents collagen IV and laminin. Each matrix significantly increased alkaline phosphatase, dipeptidyl peptidase, lactase, sucrase-isomaltase, and cell spreading in comparison to plastic. However, the basement membrane proteins collagen IV and laminin further promoted all four brush border enzymes but inhibited spreading compared to collagen I. Proliferation was most rapid on type I collagen and slowest on laminin and tissue culture plastic. Basement membrane matrix proteins may promote intestinal epithelial differentiation and inhibit proliferation compared with interstitial collagen I.