A novel marker glycoprotein for the microvillus membrane of surface colonocytes of rat large intestine and its presence in small-intestinal crypt cells

Murine mAbs were produced against purified microvillus membranes of rat colonocytes in order to establish a marker protein for this membrane. The majority of antibodies binding to the colonic microvillus membrane recognized a single protein with a mean apparent Mr of 120 kD in both proximal and distal colon samples. The antigen is membrane bound as probed by phase-partitioning studies using Triton X-114 and by the sodium carbonate extraction procedure and is extensively glycosylated as assessed by endoglycosidase F digestion. Localization studies in adult rats by light and electron microscopy revealed the microvillus membrane of surface colonocytes as the principal site of the immunoreaction. The antigen was not detectable in kidney or liver by immunoprecipitation but was present in the small intestine, where it was predominantly confined to the apical membrane of crypt cells and much less to the microvillus membrane of differentiated enterocytes. During fetal development, the antigen appears first in the colon at day 15 and 1-2 d later in the small intestine. In both segments, it initially covers the whole luminal surface but an adult-like localization pattern develops soon after birth. The antibodies were also used to develop a radiometric assay for the quantification of the antigen in subcellular fractions of colonocytes in order to assess the validity of a previously developed method for the purification of colonic brush-border membranes (Stieger, B., A. Marxer, and H.P. Hauri. 1986. J. Membr. Biol. 91:19-31.). The results suggest that we have identified a valuable marker glycoprotein for the colonic microvillus membrane, which in adult rats may also serve as a marker for early differentiation of enterocyte progenitor cells in small-intestinal crypt cells.     

SCFA increase intestinal Na absorption by induction of NHE3 in rat colon and human intestinal C2/bbe cells

Short-chain fatty acids (SCFA), produced by colonic bacterial flora fermentation of dietary carbohydrates, promote colonic Na absorption through mechanisms not well understood. We hypothesized that SCFA promote increased expression of apical membrane Na/H exchange (NHE), serving as luminal physiological cues for regulating colonic Na absorptive capacity. Studies were performed in human colonic C2/bbe (C2) monolayers and in vivo. In C2 cells exposed to butyrate, acetate, proprionate, or the poorly metabolized SCFA isobutyrate, apical membrane NHE3 activity and protein expression increased in a time- and concentration-dependent manner, whereas no changes were observed for NHE2. In contrast, no significant changes in brush-border hydrolase or villin expression were noted. Analogous to the in vitro findings, rats fed the soluble fiber pectin exhibited a time-dependent increase in colonic NHE3, but not NHE2, protein, mRNA, and brush-border activity. These changes were region-specific, as no changes were observed in the ileum. We conclude that luminal SCFA are important physiological cues for regulating colonic Na absorptive function, allowing the colon to adapt to chronic changes in dietary carbohydrate and Na loads.     

Chemoprevention of azoxymethane-induced rat aberrant crypt foci by dietary zerumbone isolated from Zingiber zerumbet.

The modifying effects of dietary feeding of zerumbone isolated from Zingiber zerumbet on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in male F344 rats. Expression of cyclooxygenase (COX)-2 in colonic mucosa exposed to AOM and/or zerumbone was also assayed. In addition, we assessed the effects of zerumbone on cell proliferation activity of crypts by counting silver-stained nucleolar organizer regions protein (AgNORs) in colonic cryptal cell nuclei. To induce ACF rats were given three weekly subcutaneous injections of AOM (15 mg/kg body weight). They were also fed the experimental diet containing 0.01% or 0.05% zerumbone for 5 weeks, starting one week before the first dosing of AOM. AOM exposure produced 84+/-13 ACF/rat at the end of the study (week 5). Dietary administration of zerumbone caused reduction in the frequency of ACF: 72+/-17 (14% reduction) at a dose of 0.01% and 45+/-18 (46% reduction, p<0.001) at a dose of 0.05%. Feeding of zerumbone significantly reduced expression of COX-2 and prostaglandins in colonic mucosa. Zerumbone feeding significantly lowered the number of AgNORs in colonic crypt cell nuclei. These findings might suggest possible chemopreventive ability of zerumbone, through suppression of COX-2 expression, cell proliferating activity of colonic mucosa, and induction of phase II detoxification enzymes in the development of carcinogen-induced ACF.     

Dietary fiber inhibits the incidence of hepatic metastasis with the anti-oxidant activity and portal scavenging functions

Dietary fiber is described as the proportion of plant foods not digested in the human small intestine. Among the various kinds of pectin, apple pectin exerts a bacteriostatic action and therefore may change the composition of the intestinal flora. The diet supplemented with 20% apple pectin significantly decreased the number and the incidence of AOM-induced colon tumors in rats. The prostaglandin E2 (PGE2) level in the distal colonic mucosa and blood of portal vein was lower in rats fed 20% pectin than those fed the basal diet. The ability of apple pectin to decrease PGE2 was dose-dependent and those results suggest an anti-inflammatory effect in the bowel. Rats fed apple pectin showed a significantly lower incidence of hepatic metastasis than those fed the basal diet. To determine whether the anti-inflammatory effect of Lactobacillus on hepatic metastasis model same as apple pectin, Lactobacillus casei was selected. Metastatic nodules were significantly reduced, especially in the group receiving pretreatment. Apple pectic oligosaccharides with small molecular weights showed highly inhibitory effects on hypoxanthin-xanthin oxidase and ferrous sulfate-hydrogen peroxide. The scavenging activity of apple pectic oligosaccharides was suitable and accelerated at high temperatures (120 degrees C, 30 min.). Apple pectin and Lactobacillus have a scavenger effect in the intestinal digestion and portal circulation system as an anti-inflammatory food and have very important function for the prevention of hepatic metastasis.     

Quantitative contribution of factors regulating rat colonic crypt epithelium: role of parenteral and enteral feeding, caloric intake, dietary cellulose level and the colon carcinogen DMH

Abstract. To elucidate the role and quantitative contribution of several exogenous factors which may regulate colon crypt mitotic activity, proliferative zone height (PZH) and crypt height, groups of rats were subjected to various feeding regimens both with and without treatment with the colon carcinogen, 1,2-dimethylhydrazine (DMH). The rats were divided into two major groups and one group was given eight weekly injections of DMH base at 9·5 mg kg^-1 body weight. Throughout this period and for two additional weeks the rats were isocalorically fed either a defined nutritionally complete diet with different levels of dietary cellulose or they were parenterally (i.v.) fed a nutritionally complete liquid formula with different caloric levels. The rats were then injected with colchicine 3 h prior to sacrifice to arrest and to collect dividing cells at metaphase. The results of multiple regression analysis of all data were interpreted to indicate that parenteral feeding caused dramatic suppression of the colon crypt height (CH) and of the number of metaphase figures per crypt (MC). Increased cellulose intake stimulated CH but suppressed MC. The CH was also stimulated by DMH. CH was positively correlated to PZH and MC. The MC was suppressed by cellulose intake and negatively correlated to PZH but was positively correlated to CH. The PZH was positively correlated to CH. These findings were related to the role of luminal food, functional workload, kcal intake and treatment with DMH on the measured colon crypt parameters. A quantitative assessment of factors that regulate the measured colonic crypt parameters was accomplished.     

Dietary Resistant Starch Alters the Characteristics of Colonic Mucosa and Exerts a Protective Effect on Trinitrobenzene Sulfonic Acid-induced Colitis in Rats

The protective effect of a dietary high-amylose cornstarch (HAS) against trinitrobenzene sulfonic acid (TNBS)-induced colitis was examined in rats. Rats were fed a HAS-free basal diet or, a 15% or 30% HAS supplemented diet for 10 d, and then received intracolonic TNBS to induce colitis and fed the respective diets for a further 8 d. HAS ingestion significantly protected colonic injuries as evidenced by lower colonic myeloperoxidase activity. Rats fed the HAS diet showed greater cecal short-chain fatty acid (SCFA) production than those fed the basal diet. Further, just before TNBS administration, HAS ingestion dose-dependently increased fecal and cecal mucin contents, and protein and nucleic acid contents in the colonic mucosa. HAS ingestion also reduced colonic permeability. The protective effect of HAS ingestion on TNBS-induced colitis is perhaps exerted through alterations in colonic mucosa, possibly due to cecal SCFA production.     

Quantitative contribution of factors regulating rat colonic crypt epithelium: role of parenteral and enteral feeding, caloric intake, dietary cellulose level and the colon carcinogen DMH

To elucidate the role and quantitative contribution of several exogenous factors which may regulate colon crypt mitotic activity, proliferative zone height (PZH) and crypt height, groups of rats were subjected to various feeding regimens both with and without treatment with the colon carcinogen, 1,2-dimethylhydrazine (DMH). The rats were divided into two major groups and one group was given eight weekly injections of DMH base at 9.5 mg kg-1 body weight. Throughout this period and for two additional weeks the rats were isocalorically fed either a defined nutritionally complete diet with different levels of dietary cellulose or they were parenterally (i.v.) fed a nutritionally complete liquid formula with different caloric levels. The rats were then injected with colchicine 3 h prior to sacrifice to arrest and to collect dividing cells at metaphase. The results of multiple regression analysis of all data were interpreted to indicate that parenteral feeding caused dramatic suppression of the colon crypt height (CH) and of the number of metaphase figures per crypt (MC). Increased cellulose intake stimulated CH but suppressed MC. The CH was also stimulated by DMH. CH was positively correlated to PZH and MC. The MC was suppressed by cellulose intake and negatively correlated to PZH but was positively correlated to CH. The PZH was positively correlated to CH. These findings were related to the role of luminal food, functional workload, kcal intake and treatment with DMH on the measured colon crypt parameters. A quantitative assessment of factors that regulate the measured colonic crypt parameters was accomplished.     

Dietary resistant starch alters the characteristics of colonic mucosa and exerts a protective effect on trinitrobenzene sulfonic acid-induced colitis in rats

The protective effect of a dietary high-amylose cornstarch (HAS) against trinitrobenzene sulfonic acid (TNBS)-induced colitis was examined in rats. Rats were fed a HAS-free basal diet or, a 15% or 30% HAS supplemented diet for 10 d, and then received intracolonic TNBS to induce colitis and fed the respective diets for a further 8 d. HAS ingestion significantly protected colonic injuries as evidenced by lower colonic myeloperoxidase activity. Rats fed the HAS diet showed greater cecal short-chain fatty acid (SCFA) production than those fed the basal diet. Further, just before TNBS administration, HAS ingestion dose-dependently increased fecal and cecal mucin contents, and protein and nucleic acid contents in the colonic mucosa. HAS ingestion also reduced colonic permeability. The protective effect of HAS ingestion on TNBS-induced colitis is perhaps exerted through alterations in colonic mucosa, possibly due to cecal SCFA production.     

Pericryptal Myofibroblast Growth in Rat Descending Colon Induced by Low-Sodium Diets Is Mediated by Aldosterone and not by Angiotensin II

Pericryptal myofibroblast growth in descending colonic crypts correlates with the activation of the renin-angiotensin-aldosterone system. Earlier work showed that during the transition from a high-Na⁺ (HS) to low-Na⁺ (LS) diet there are changes in the colonic crypt wall and pericryptal sheath. As LS diet increases both aldosterone and angiotensin II, the aim here was to determine their individual contributions to the trophic changes in colonic crypts. Experiments were conducted on control and adrenalectomized Sprague-Dawley rats fed an HS diet and then switched to LS diet for 3 days and supplemented with aldosterone or angiotensin II. The actions of the angiotensin-converting enzyme inhibitor captopril, the angiotensin receptor antagonist losartan and the aldosterone antagonist spironolactone on extracellular matrix proteins, claudin 4 and E-cadherin myofibroblast proteins, α-smooth muscle actin (α-SMA) and OB-cadherin (cadherin 11), angiotensin type 1 and TGFβr1 membrane receptors were determined by immunolocalization in fixed distal colonic mucosa. The LS diet or aldosterone supplementation following ADX in HS or LS increased extracellular matrix, membrane receptors and myofibroblast proteins, but angiotensin alone had no trophic effect on α-SMA. These results show that aldosterone stimulates myofibroblast growth in the distal colon independently of dietary Na⁺ intake and of angiotensin levels. This stimulus could be a genomic response or secondary to stretch of the pericryptal sheath myofibroblasts accompanying enhanced rates of crypt fluid absorption.     

Interactive Effects of Indigestible Carbohydrates,Protein Type,and Protein Level on Biomarkers of Large Intestine Health in Rats

The effects of indigestible carbohydrates, protein type, and protein level on large intestine health were examined in rats. For 21 days, 12 groups of six 12-week-old male Wistar rats were fed diets with casein (CAS), or potato protein concentrate (PPC), providing 14% (lower protein level; LP), or 20% (higher protein level; HP) protein, and containing cellulose, resistant potato starch, or pectin. Fermentation end-products, pH, and β-glucuronidase levels in cecal digesta, and ammonia levels in colonic digesta were determined. Cecal digesta, tissue weights, cecal and colon morphology, and colonocyte DNA damage were also analyzed. Digesta pH was lower, whereas relative mass of cecal tissue and digesta were higher in rats fed pectin diets than in those fed cellulose. Cecal parameters were greater in rats fed PPC and HP diets than in those fed CAS and LP diets, respectively. Short-chain fatty acid (SCFA) concentrations were unaffected by protein or carbohydrate type. Total SCFA, acetic acid, and propionic acid concentrations were greater in rats fed LP diets than in those fed HP. Cecal pool of isobutyric and isovaleric acids was greater in rats fed PPC than in those fed CAS diets. PPC diets decreased phenol concentration and increased ammonia concentration in cecal and colonic digesta, respectively. Cecal crypt depth was greater in rats fed PPC and HP diets, and was unaffected by carbohydrates; whereas colonic crypt depth was greater in rats fed cellulose. Myenteron thickness in the cecum was unaffected by nutrition, but was greater in the colon of rats fed cellulose. Colonocyte DNA damage was greater in rats fed LP diets than in those fed HP diets, and was unaffected by carbohydrate or protein type. It was found that nutritional factors decreasing cecal digesta weight contribute to greater phenol production, increased DNA damage, and reduced ammonia concentration in the colon.     

Dietary supplementation with ovine serum immunoglobulin is associated with an increased gut luminal mucin concentration in the growing rat

The mucus layer covering the gut epithelium is pivotal to host defence and is affected by various dietary components. Part of the reported beneficial effect of dietary immunoglobulins (Igs) on gut health may be due to effects on the gut mucus layer. The aim was to determine whether orally administered ovine serum Ig influence goblet cell count, mucin gene expression and digesta mucin protein content in the gut of the growing rat. Fourteen Sprague-Dawley male growing rats were used in a 21-day study and were fed either a casein-based control diet (CON; no Ig) or a similar diet but containing freeze-dried ovine Ig (FDOI). Daily food intake and growth rate were not affected by the dietary treatments. When compared to the rats consuming CON diet, those consuming the FDOI diet had significantly (P < 0.05) more intact and cavitated goblet cells in the intestinal villi. A similar result was found for crypt goblet cells in the small intestine and colon. Ileal Muc2, Muc3, Muc4 and stomach Muc5Ac mRNA expressions for the FDOI animals were higher (P < 0.05) compared to the the CON animals. Mucin protein content was higher (P < 0.05) in the stomach, ileum and colonic digesta of rats fed the FDOI diet. In conclusion, orally administered FDOI influenced gut mucins in the growing rat as evidenced by increased mucin gene expression and digesta mucin protein concentrations as well as an increased goblet cell count.     

Expression of the peptide transporter hPepT1 in human colon: a potential route for colonic protein nitrogen and drug absorption

Substrates of the proton-coupled peptide transporter, hPepT1, include dietary di- and tripeptides plus therapeutically important drugs such as the beta-lactam antibiotics and angiotensin-converting enzyme inhibitors. Expression and function of hPepT1 in the small bowel is well established. We have compared levels of hPepT1 mRNA expression in regions of human gut by RT-PCR methods and examined the expression of hPepT1 in normal human colon using an anti-hPepT1 antipeptide antibody. hPepT1 mRNA was expressed in the large intestine, although at lower levels than in the small intestine. Quantitatively, expression in ileum was 4.6-fold greater than in sigmoid colon. Immunoreactive hPepT1 was detected in human colon at lower levels than in ileum. The pattern of expression differed between the two tissues: whilst expression in the ileum was localised to the apical enterocyte membrane along the length of the crypt-villus axis, expression in the colonocyte was detected at the apical membrane towards the luminal surface but predominantly at the basal membrane towards the base of the crypt. We conclude that distal regions of the bowel express hPepT1, which may provide a mechanism for colonic protein-nitrogen absorption and for absorption of therapeutically important peptidomimetic drugs.     

Isolation of pig colonic crypts for cytotoxic assay of luminal compounds: effects of hydrogen sulfide,ammonia, and deoxycholic acid

Some colonic luminal molecules resulting from bacterial metabolism of alimentary or endogenous compounds are believed to exert various effects on the colonic epithelial cell physiology. We isolated surface epithelial cells and intact colonic crypts in order to test bacterial metabolites in the pig model, which is often considered relevant for extrapolation to the physiopathology of the human gastrointestinal tract. Using colonocytes isolated with EDTA, we found that the initial cell viability, estimated by the membrane integrity and oxidative capacity measurement, fell rapidly despite several experimental attempts to preserve it such as the use of a medium designed to increase the adherence of epithelial cells and of a coated extracellular matrix, the presence in the culture medium of the oxidative substrate butyrate, and the use of an inhibitor of the caspases involved in cell apoptosis. In contrast, using dispase and collagenase as proteolytic agents, we were able to obtain pig colonic crypts that maintain an excellent membrane integrity after 4 h. Using this preparation, we were able to test the presumably cytotoxic luminal compounds hydrogen sulfide, ammonia, and deoxycholic acid on colonic crypt viability. Of these, only deoxycholic acid was found to significantly alter the cellular membrane integrity. It is concluded that pig colonic crypts can be useful for thein vitro appraisal of the cytotoxic properties of luminal compounds. This revised version was published online in July 2006 with corrections to the Cover Date.     

Suppressive effect of excess dietary histidine on the expression of hepatic metallothionein-1 in rats

The gene expression of liver metallothionein-1 in excess dietary histidine was investigated by feeding rats ad libitum on either a basal or histidine-excess (50 g of L-histidine per kg of diet) diet for 5 d. The copper content of the liver and zinc level in the serum of the rats fed on the histidine-excess diet were lower by 21% and 61%, respectively of the figures for the rats fed on the basal diet, but the zinc content of the liver and copper level in the serum were not affected. Excess dietary histidine caused an increase in the urinary output of both copper and zinc. The level of liver metallothionine-1 mRNA was markedly lower at 19% in the rats fed on the a histidine-excess diet compared to the level with the basal diet. It thus appears that such a response by the level of liver metallothionein-1 mRNA might have been be due to the lower content of liver copper.     

Plasma cholesterol-lowering effect on rats of dietary fiber extracted from immature plants

Crude dietary fiber samples were prepared from beet, cabbage, Japanese radish, onion and mung bean sprouts (BF, CF, RF, OF and MF, respectively). These samples contained total dietary fiber at the levels of 814, 699, 760, 693 and 666 g/kg, respectively. To examine the effect of these dietary fiber sources on the plasma cholesterol concentration, male Sprague-Dawley rats were fed on a fiber-free (FF) diet or on an FF diet supplemented with 5% or 10% dietary fiber. Dietary fiber extracted from vegetables, wood cellulose (CL), pectin (PE) and guar gum (GG) were used as the fiber sources. Compared with the rats fed on the FF diet, a significant reduction in the plasma cholesterol concentration was observed in the rats fed on BF, CF, RF, MF, PE or GG after a 21-d feeding period. Cecal acetate, n-butyrate and total short-chain fatty acids were significantly higher in the rats fed on these dietary fibers, except for CF, than in those fed on the FF diet. A negative correlation was apparent between the total dietary fiber content, hemicellulose content and pectin content of each dietary fiber source and the plasma cholesterol concentration. These results suggest that some vegetable fibers exert a plasma cholesterol-lowering effect through cecal fermentation of these fibers.     

Effect of pectin on the function of the rat exocrine pancreas

The author studied the effect of three weeks' administration of a 5% pectin concentration in the standard (Larsen) diet on basal and cholecystokinin octapeptide-(CCK8-) stimulated pancreatic exosecretion in rats. Protein, amylase and trypsinogen output determined in pancreatic juice obtained under basal conditions and after CCK8 stimulation of the exocrine pancreas showed no statistically significant differences between rats fed on the standard and the pectin-enriched Larsen diet. Our observations and studies by other authors show that the direct effect of dietary fibre on the exocrine function of the pancreas is an open question as far as further research is concerned.