Morphometric study of the small intestinal mucosa in young, adult, and old rats submitted to protein deficiency and rehabilitation

Linear and stereological morphometric methods were applied to the jejunal and ileal mucosa of young, adult, and old male Wistar rats submitted to protein deficiency and rehabilitation. The animals were fed ad libitum a 2% casein diet during 42 days and then received a 20% casein diet for 30 days. Food intake, body weights, and plasma protein concentrations were recorded. In the young protein deficient rats values of mucosal height, surface area, and volume of the lamina propria were significantly lower than those of their age controls in both jejunum and ileum. In adults the differences were less marked and in the old rats all parameters were found to be unaltered by the protein deficient diet. The surface-to-volume ratio showed no significant differences between control and protein deficient in all three age groups, meaning that villus pattern did not change with protein deficiency. On rehabilitation, a striking difference between jejunum and ileum was observed in the young rats; all parameters returned to control levels in the jejunum, while they remained lower than those of their controls in the ileum.

A comparison of mucosal surface area and villous histology in small intestines of the brazilian free-tailed bat (Tadarida brasiliensis) and the mouse (Mus musculus)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Gastric and small intestinal lesions after partial stomach resection with Billroth II or Roux-en-Y reconstruction in the rat

The evolution and phenotypic expression of mucosal lesions of the gastric stump were investigated in male rats submitted to gastric resection with reconstruction by the Billroth II technique (BII with biliopancreatic reflux, BPR) or by the Roux-en-Y procedure (without BPR). Animals were studied at 24, 36, 54 and 64 weeks after surgery and the phenotypic expression of lesions analysed using routine hematoxylin and eosin staining, immunohistochemical staining for pepsinogen isoenzyme 1 and histochemical procedures for mucins (paradoxical concanavalin A, galactose oxidase Schiff (GOS) and sialidase GOS reactions). BPR was found to be responsible for the formation of adenomatous hyperplasia (AH), increasing in incidence and size with time, since the Roux-en-Y procedure failed to induce the gastric stump lesions observed after BII reconstruction. AHs always occurred in the transition of the gastrojejunal junction, a site offering special conditions for BPR influence, and were classified as gastric (G), intestinal (I) and G+I types according to their phenotypic expression. No pure I type AH was diagnosed at any time point. The G and G+I types developed at approximately equal incidences (i.e., G type 7/17, G+I type 10/17 at the 64th week). It was suggested that both gastric and intestinal mucosal elements were stimulated to proliferate by BPR, with the gastric mucosa tending to demonstrate AH. Intestinal type components of AH were found adjacent to the jejunum and not at the stomach margin, indicating an origin from intestinal mucosa. No metaplasia of the gastric mucosa was observed in any animal after partial gastric resection. In 101 rats submitted to the BII procedure, 5 mucinous adenocarcinomas were eventually diagnosed, mostly located in the subserosa of the gastrojejunal junction. All carcinomas expressed the phenotype of cells of the small intestine. Evidence of malignant transformation within the gastric components of AH was not observed even at the 64th week. In conclusion, all lesions induced by BPR in the rat remnant stomach are benign, and the few true cancers that arise in association are derived from the small intestine.

Cholesteryl ester accumulation and accelerated cholesterol absorption in intestine-specific hormone sensitive lipase-null mice

Hormone sensitive lipase (HSL) regulates the hydrolysis of acylglycerols and cholesteryl esters (CE) in various cells and organs, including enterocytes of the small intestine. The physiological role of this enzyme in enterocytes, however, stayed elusive. In the present study we generated mice lacking HSL exclusively in the small intestine (HSLiKO) to investigate the impact of HSL deficiency on intestinal lipid metabolism and the consequences on whole body lipid homeostasis. Chow diet-fed HSLiKO mice showed unchanged plasma lipid concentrations. In addition, feeding with high fat/high cholesterol (HF/HC) diet led to unaltered triglyceride but increased plasma cholesterol concentrations and CE accumulation in the small intestine. The same effect was observed after an acute cholesterol load. Gavaging of radioactively labeled cholesterol resulted in increased abundance of radioactivity in plasma, liver and small intestine of HSLiKO mice 4h post-gavaging. However, cholesterol absorption determined by the fecal dual-isotope ratio method revealed no significant difference, suggesting that HSLiKO mice take up the same amount of cholesterol but in an accelerated manner. mRNA expression levels of genes involved in intestinal cholesterol transport and esterification were unchanged but we observed downregulation of HMG-CoA reductase and synthase and consequently less intestinal cholesterol biosynthesis. Taken together our study demonstrates that the lack of intestinal HSL leads to CE accumulation in the small intestine, accelerated cholesterol absorption and decreased cholesterol biosynthesis, indicating that HSL plays an important role in intestinal cholesterol homeostasis.

Evidence of native starch degradation with human small intestinal maltase-glucoamylase (recombinant)

Action of human small intestinal brush border carbohydrate digesting enzymes is thought to involve only final hydrolysis reactions of oligosaccharides to monosaccharides. In vitro starch digestibility assays use fungal amyloglucosidase to provide this function. In this study, recombinant N-terminal subunit enzyme of human small intestinal maltase-glucoamylase (rhMGAM-N) was used to explore digestion of native starches from different botanical sources. The susceptibilities to enzyme hydrolysis varied among the starches. The rate and extent of hydrolysis of amylomaize-5 and amylomaize-7 into glucose were greater than for other starches. Such was not observed with fungal amyloglucosidase or pancreatic alpha-amylase. The degradation of native starch granules showed a surface furrowed pattern in random, radial, or tree-like arrangements that differed substantially from the erosion patterns of amyloglucosidase or alpha-amylase. The evidence of raw starch granule degradation with rhMGAM-N indicates that pancreatic alpha-amylase hydrolysis is not a requirement for native starch digestion in the human small intestine.

Compartmentalised expression of meprin in small intestinal mucosa: enhanced expression in lamina propria in coeliac disease

Epithelial cells in the human small intestine express meprin, an astacin-like metalloprotease, which accumulates normally at the brush border membrane and in the gut lumen. Therefore, meprin is targeted towards luminal components. In coeliac disease patients, peptides from ingested cereals trigger mucosal inflammation in the small intestine, disrupting epithelial cell differentiation and function. Using in situ hybridisation on duodenal tissue sections, we observed a marked shift of meprin mRNA expression from epithelial cells, the predominant expression site in normal mucosa, to lamina propria leukocytes in coeliac disease. Meprin thereby gains access to the substrate repertoire present beneath the epithelium.

A stereological comparison of villous and microvillous surfaces in small intestines of frugivorous and entomophagous bats: species, inter-individual and craniocaudal differences

The extents of functional surfaces (villi, microvilli) have been estimated at different longitudinal sites, and in the entire small intestine, for three species of bats belonging to two feeding groups: insect- and fruit-eaters. In all species, surface areas and other structural quantities tended to be greatest at more cranial sites and to decline caudally. The entomophagous bat (Miniopterus inflatus) had a mean body mass (coefficient of variation) of 8.9 g (5%) and a mean intestinal length of 20 cm (6%). The surface area of the basic intestinal tube (primary mucosa) was 9.1 cm2 (10%) but this was amplified to 48 cm2 (13%) by villi and to 0.13 m2 (20%) by microvilli. The total number of microvilli per intestine was 4 x 10(11) (20%). The average microvillus had a diameter of 8 nm (10%), a length of 1.1 microns (22%) and a membrane surface area of 0.32 micron 2 (31%). In two species of fruit bats (Epomophorus wahlbergi and Lisonycteris angolensis), body masses were greater and intestines longer, the values being 76.0 g (18%) and 76.9 g (4%), and 73 cm (16%) and 72 cm (7%), respectively. Surface areas were also greater, amounting to 76 cm2 (26%) and 45 cm2 (8%) for the primary mucosa, 547 cm2 (29%) and 314 cm2 (16%) for villi and 2.7 m2 (23%) and 1.5 m2 (18%) for microvilli. An increase in the number of microvilli, 33 x 10(11) (19%) and 15 x 10(11) (24%) per intestine, contributed to the more extensive surface area but there were concomitant changes in the dimensions of microvilli. Mean diameters were 94 nm (8%) and 111 nm (4%), and mean lengths were 2.8 microns (12%) and 2.9 microns (10%), respectively. Thus, an increase in the surface area of the average microvillus to 0.83 micron 2 (12%) and 1.02 microns 2 (11%) also contributed to the greater total surface area of microvilli. The lifestyle-related differences in total microvillous surface areas persisted when structural quantities were normalised for the differences in body masses. The values for total microvillous surface area were 148 cm2g-1 (20%) in the entomophagous bat, 355 cm2g-1 (20%) in E. wahlbergi and 192 cm2g-1 (17%) in L. angolensis. This was true despite the fact that the insecteater possessed a greater length of intestine per unit of body mass: 22 mm g-1 (8%) versus 9-10 mm g-1 (9-10%) for the fruit-eaters.

Effect of indomethacin on bile acid-phospholipid interactions: implication for small intestinal injury induced by nonsteroidal anti-inflammatory drugs.

The injurious effect of nonsteroidal anti-inflammatory drugs (NSAIDs) in the small intestine was not appreciated until the widespread use of capsule endoscopy. Animal studies found that NSAID-induced small intestinal injury depends on the ability of these drugs to be secreted into the bile. Because the individual toxicity of amphiphilic bile acids and NSAIDs directly correlates with their interactions with phospholipid membranes, we propose that the presence of both NSAIDs and bile acids alters their individual physicochemical properties and enhances the disruptive effect on cell membranes and overall cytotoxicity. We utilized in vitro gastric AGS and intestinal IEC-6 cells and found that combinations of bile acid, deoxycholic acid (DC), taurodeoxycholic acid, glycodeoxycholic acid, and the NSAID indomethacin (Indo) significantly increased cell plasma membrane permeability and became more cytotoxic than these agents alone. We confirmed this finding by measuring liposome permeability and intramembrane packing in synthetic model membranes exposed to DC, Indo, or combinations of both agents. By measuring physicochemical parameters, such as fluorescence resonance energy transfer and membrane surface charge, we found that Indo associated with phosphatidylcholine and promoted the molecular aggregation of DC and potential formation of larger and isolated bile acid complexes within either biomembranes or bile acid-lipid mixed micelles, which leads to membrane disruption. In this study, we demonstrated increased cytotoxicity of combinations of bile acid and NSAID and provided a molecular mechanism for the observed toxicity. This mechanism potentially contributes to the NSAID-induced injury in the small bowel.

Efficient and Safe Small-Bowel Adhesiolysis

Small-intestine adhesiolysis can be very time consuming and may be associated with bowel wall damage. The risk for injuries to the small or large bowel resulting in increased morbidity and costs is considerable. Both efficient and gentle dissection of adhesions is important in order to avoid intraoperative perforation or, worse, postoperative intestinal leaks. We present a technique using drops of body-warm isotonic saline solution to create an edematous swelling of the adhesions. This procedure not only protects the bowel from cooling and drying, but also simplifies the dissection and, thus, lowers the risk of intestinal lesions.

Binding studies on isolated porcine small intestinal mucosa and in vitro toxicity studies reveal lack of effect of C. perfringens beta-toxin on the porcine intestinal epithelium

Beta-toxin (CPB) is the essential virulence factor of C. perfringens type C causing necrotizing enteritis (NE) in different hosts. Using a pig infection model, we showed that CPB targets small intestinal endothelial cells. Its effect on the porcine intestinal epithelium, however, could not be adequately investigated by this approach. Using porcine neonatal jejunal explants and cryosections, we performed in situ binding studies with CPB. We confirmed binding of CPB to endothelial but could not detect binding to epithelial cells. In contrast, the intact epithelial layer inhibited CPB penetration into deeper intestinal layers. CPB failed to induce cytopathic effects in cultured polarized porcine intestinal epithelial cells (IPEC-J2) and primary jejunal epithelial cells. C. perfringens type C culture supernatants were toxic for cell cultures. This, however, was not inhibited by CPB neutralization. Our results show that, in the porcine small intestine, CPB primarily targets endothelial cells and does not bind to epithelial cells. An intact intestinal epithelial layer prevents CPB diffusion into underlying tissue and CPB alone does not cause direct damage to intestinal epithelial cells. Additional factors might be involved in the early epithelial damage which is needed for CPB diffusion towards its endothelial targets in the small intestine.

The role of nitric oxide in small intestinal motility in a model of acute inflammation

Motility responses of the small intestine of iNOS deficient mice (iNOS −/−) and their wildtype littermates (iNOS+/+) to the inflammatory challenge of lipopolysaccharide (LPS) were investigated. LPS administration failed to attenuate intestinal transit in iNOS−/− mice but depressed transit in their iNOS+/+ littermates. Supporting an inhibitory role for sustained nitric oxide (NO) synthesis in the regulation of intestinal motility during inflammation, iNOS immunoreactivity was upregulated in all regions of the small intestine of iNOS+/+ mice. In contrast, neuronal NOS was barely affected. Cyclooxygenase activation was determined by prostaglandin E2 (PGE2) concentration. Following LPS challenge, PGE2 levels were elevated in all intestinal segments in both animal groups. Moreover, COX-1 and COX-2 protein levels were elevated in iNOS+/+ mice in response to LPS, while COX-2 levels were similarly increased in iNOS −/− intestine. However, no apparent relationship was observed between increased prostaglandin concentrations and attenuated intestinal transit. The presence of heme oxygenase 1 (HO-1) in the murine small intestine was also investigated. In both animal groups HO-1 immunoreactivity in the proximal intestine increased in response to treatment, while the constitutive protein levels detected in the middle and distal intestine were unresponsive to LPS administration. No apparent correlation of HO-1 to the suppression of small intestinal motility induced by LPS administration was detected. The presence of S-nitrosylated contractile proteins in the small intestine was determined. γ-smooth muscle actin was basally nitrosylated as well as in response to LPS, but myosin light chain kinase and myosin regulatory chain (MLC20) were not. In conclusion, in a model of acute intestinal inflammation, iNOS-produced NO plays a significant role in suppressing small intestinal motility while nNOS, COX-1, COX-2 and HO-1 do not participate in this event. S-nitrosylation of γ-smooth muscle actin is associated with elevated levels of nitric oxide in the smooth muscle of murine small intestine. ^

Macrophage inflammatory protein-2: Chromosomal regulation in rat small intestinal epithelial cells

Nonpathogenic, resident bacteria participate in the pathogenesis of inflammation in the small intestine, but the molecular messages produced by such bacteria are unknown. Inflammatory responses involve the recruitment of specific leukocyte subsets. We, therefore, hypothesized that butyrate, a normal bacterial metabolite, may modulate chemokine secretion by epithelial cells, by amplifying their response to proinflammatory signals. We studied the expression of the chemokine, macrophage inflammatory protein-2 (MIP-2) by the rat small intestinal epithelial cell line, IEC-6. Cells were stimulated with lipopolysaccharide or with interleukin 1β (IL-1β) and incubated with sodium butyrate. Acetylation of histones was examined in Triton X acetic acid–urea gels by PAGE. Unstimulated IEC-6 cells did not secrete MIP-2. However, lipopolysaccharide and IL-1β induced MIP-2 expression. Butyrate enhanced MIP-2 secretion both in lipopolysaccharide-stimulated and IL-1β-stimulated enterocytes; but butyrate alone did not induce MIP-2 expression. Butyrate increased the acetylation of histones extracted from the nuclei of IEC-6 cells. Furthermore, acetylation of histones (induced by trichostatin A, a specific inhibitor of histone deacetylase) enhanced MIP-2 expression by cells stimulated with IL-1β. In conclusion, trichostatin A reproduced the effects of butyrate on MIP-2 secretion. Butyrate, therefore, increases MIP-2 secretion in stimulated cells by increasing histone acetylation. We speculate that butyrate carries information from bacteria to epithelial cells. Epithelial cells transduce this signal through histone deacetylase, modulating the secretion of chemokines.

Galectin-4 and Small Intestinal Brush Border Enzymes Form Clusters

Detergent-insoluble complexes prepared from pig small intestine are highly enriched in several transmembrane brush border enzymes including aminopeptidase N and sucrase-isomaltase, indicating that they reside in a glycolipid-rich environment in vivo. In the present work galectin-4, an animal lectin lacking a N-terminal signal peptide for membrane translocation, was discovered in these complexes as well, and in gradient centrifugation brush border enzymes and galectin-4 formed distinct soluble high molecular weight clusters. Immunoperoxidase cytochemistry and immunogold electron microscopy showed that galectin-4 is indeed an intestinal brush border protein; we also localized galectin-4 throughout the cell, mainly associated with membraneous structures, including small vesicles, and to the rootlets of microvillar actin filaments. This was confirmed by subcellular fractionation, showing about half the amount of galectin-4 to be in the microvillar fraction, the rest being associated with insoluble intracellular structures. A direct association between the lectin and aminopeptidase N was evidenced by a colocalization along microvilli in double immunogold labeling and by the ability of an antibody to galectin-4 to coimmunoprecipitate aminopeptidase N and sucrase-isomaltase. Furthermore, galectin-4 was released from microvillar, right-side-out vesicles as well as from mucosal explants by a brief wash with 100 mM lactose, confirming its extracellular localization. Galectin-4 is therefore secreted by a nonclassical pathway, and the brush border enzymes represent a novel class of natural ligands for a member of the galectin family. Newly synthesized galectin-4 is rapidly “trapped” by association with intracellular structures prior to its apical secretion, but once externalized, association with brush border enzymes prevents it from being released from the enterocyte into the intestinal lumen.

Generation of in vivo activating factors in the ischemic intestine by pancreatic enzymes

One of the early events in physiological shock is the generation of activators for leukocytes, endothelial cells, and other cells in the cardiovascular system. The mechanism by which these activators are produced has remained unresolved. We examine here the hypothesis that pancreatic digestive enzymes in the ischemic intestine may be involved in the generation of activators during intestinal ischemia. The lumen of the small intestine of rats was continuously perfused with saline containing a broadly acting pancreatic enzyme inhibitor (6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfate, 0.37 mM) before and during ischemia of the small intestine by splanchnic artery occlusion. This procedure inhibited activation of circulating leukocytes during occlusion and reperfusion. It also prevented the appearance of activators in portal venous and systemic artery plasma and attenuated initiating symptoms of multiple organ injury in shock. Intestinal tissue produces only low levels of activators in the absence of pancreatic enzymes, whereas in the presence of enzymes, activators are produced in a concentration- and time-dependent fashion. The results indicate that pancreatic digestive enzymes in the ischemic intestine serve as an important source for cell activation and inflammation, as well as multiple organ failure.

Genetic engineering of carbohydrate biosynthetic pathways in transgenic mice demonstrates cell cycle-associated regulation of glycoconjugate production in small intestinal epithelial cells.

Proliferation, migration-associated differentiation, and cell death occur continuously and in a spatially well-organized fashion along the crypt-villus axis of the mouse small intestine, making it an attractive system for studying how these processes are regulated and interrelated. A pathway for producing glycoconjugates was engineered in adult FVB/N transgenic mice by expressing a human alpha 1,3/4-fucosyltransferase (alpha 1,3/4-FT; EC 2.4.1.65) along the length of this crypt-villus axis. The alpha 1,3/4-FT can use lacto-N-tetraose or lacto-neo-N-tetraose core chains to generate Lewis (Le) blood group antigens Le(a) or Le(x), respectively, and H type 1 or H type 2 core chains to produce Leb and Le(y). Single- and multilabel immunohistochemical studies revealed that expression of the alpha 1,3/4-FT results in production of Le(a) and Leb antigens in both undifferentiated proliferated crypt cells and in differentiated postmitotic villus-associated epithelial cells. In contrast, Le(x) antigens were restricted to crypt cells. Villus enterocytes can be induced to reenter the cell cycle by expression of simian virus 40 tumor antigen under the control of a promoter that only functions in differentiated members of this lineage. Bitransgenic animals, generated from a cross of FVB/N alpha 1,3/4-FT with FVB/N simian virus 40 tumor antigen mice, expand the range of Le(x) expression to include villus-associated enterocytes that have reentered the cell cycle. Thus, the fucosylations unveil a proliferation-dependent switch in oligosaccharide production, as defined by a monoclonal antibody specific for the Le(x) epitope. These findings show that genetic engineering of oligosaccharide biosynthetic pathways can be used to define markers for entry into, or progression through, the cell cycle and to identify changes in endogenous carbohydrate metabolism that occur when proliferative status is altered in a manner that is not deleterious to the system under study.