Jejunal Explants Research Articles

Insulin decreases chylomicron production in human fetal small intestine

The objective of this study was to examine the effect of insulin on lipoprotein synthesis and secretion in human fetal intestine. Jejunal explants were cultured with [ 14C]oleic acid in Leibovitz medium for 42 h. Although the addition of insulin (30 U) did not alter the incorporation of [ 14C]oleic acid into triglycerides, phospholids and cholesteryl esters in the tissue, it significantly decreased ( P < 0.05) the level of triglycerides (20%) in the medium. Among the three lipoprotein classes (chylomicron, VLDL, and HDL) isolated by ultracentrifugation, the chylomicron level was found significantly ( P < 0.05) diminished in the medium (29%). Neither the lipid chemical composition of CM or that of VLDL, LDL and HDL was affected by the presence of insulin. These results suggest that chylomicron synthesis is modulated by insulin, whereas lipoprotein distribution and lipid composition are not regulated by this hormone.

Apolipoprotein synthesis in newborn piglet intestinal explants.

To determine the effects of hormones and epidermal growth factor (EGF) on the small intestinal synthesis of apolipoproteins B-48, A-I, and A-IV in the neonatal mammal, apolipoprotein synthesis by proximal jejunal explants from 2-d-old female piglets was studied in tissue culture. Initial comparison studies with various media showed optimal total protein and apo A-I synthesis with Williams' medium E without fetal bovine serum. Sets of explants were prepared containing EGF and various hormones in the medium. After 35S-methionine radiolabeling, explants were homogenized, and specific apolipoprotein synthesis was quantitated by immunoprecipitation as the percentage of total protein synthesis. Apo B-48 synthesis was not affected by any additives except the combination of EGF and hydrocortisone, which slightly decreased synthesis. Apo A-I synthesis was significantly increased by EGF. This EGF-induced increase in apo A-I synthesis was blunted by concomitant treatment with hydrocortisone. In contrast, the combination of insulin and hydrocortisone induced a significant increase in apo A-I synthesis. Although EGF and insulin modestly increased apo A-IV synthesis, the combination of insulin and hydrocortisone treatment up-regulated apo A-IV synthesis by 2.6-fold. Thyroid hormone lacked effect on synthesis of any of the apolipoproteins. EGF, glucocorticoids, and insulin may play regulatory roles in the developmental expression of apolipoprotein synthesis in the neonatal small intestine.

Intestinal lipids and lipoproteins in the human fetus: modulation by epidermal growth factor.

The aim of the present investigation was first, to examine the ability of human fetal intestine (17-20 wk) to incorporate fatty acid into esterified lipids; and second, to study in vitro lipoprotein synthesis and secretion by fetal explants, as well as the effect of epidermal growth factor (EGF) on these processes. Cultured fetal jejunal explants were incubated in Leibovitz medium for 42 h with [14C]oleate. Both triglycerides (TG) and phospholipids (PL) were the major labeled products. Whereas TG were predominant (80%) in the culture medium, PL accounted for more than 50% of total tissue lipids. More than 60% of the radioactivity in PL was associated with phosphatidylcholine. Some labeling (< 5%) was also recovered in the cholesteryl ester fraction. Active exocytosis was demonstrated by the accumulation of newly synthesized esterified lipids in the medium and the presence of lipoproteins in the basolateral membrane region and intercellular spaces. Most of the newly synthesized lipids were found in lipoproteins of d < 0.97 g/ml (51.2%) and d < 1.21 g/ml (39.3%), whereas the rest were recovered in d < 1.006 g/ml (9.8%) and 1.063 g/ml (5.6%). A similar trend characterized the lipoprotein secretion. The synthesis of the d < 0.97 g/ml fraction (30,653 +/- 4,122 dpm/mg protein) was significantly greater than the 1.006 g/ml fraction (5,897 +/- 1,734), P < 0.005. The secretion of d < 0.97 g/ml particles into the medium was also five fold higher than that of the d < 1.006 g/ml fraction (P < 0.01). The addition of EGF to the culture medium (25, 50, and 100 ng/ml) significantly enhanced the d < 0.97 g/ml lipoprotein secretion (25-40%) and decreased the d 1.006 g/ml and 1.063 g/ml fraction output. The lipid composition of these lipoprotein fractions was never altered by the presence of EGF, suggesting that the number of lipoprotein particles, rather than size, was modified by the growth factor. The present findings provide the first evidence that the human fetal intestine has the capacity to elaborate lipoprotein fractions for the transport of newly synthesized lipids. Furthermore, our data suggest that EGF, present in significant quantity in saliva, amniotic fluid, and bile, can modulate the release of TG-rich lipoproteins by fetal intestinal explants.

The expression of lactase enzymatic activity and mRNA in human fetal jejunum Effect of organ culture and of treatment with hydrocortisone

Very sensitive procedures were developed for the parallel determination of intestinal lactase (LPH) activity and the cognate mRNA. Between 14 and 20 weeks of gestation, lactase activity is low and varies only slightly; at 37 weeks, a relatively high level of activity is observed. The amounts of LPH mRNA correlates with the enzymatic activity ( r = 0.64). Culture of fetal jejunal explants for 5 days induces by itself a 2-fold increase in LPH mRNA, without any significant change in lactase enzymatic activity. This increase may reflect the loss of a negative transcriptional regulation operative in vivo, and suggests all additional post-transcriptional regulatory component. The addition of hydrocortisone (50 ng/ml) during culture induces a doubling of lactase activity without variation in LHP mRNA, indicating a post-transcriptional modulation by hydrocortisone. The intestinal lysosomal acid β-galactosidase activity was shown to be unaffected by hydrocortisone treatment. This observation clearly illustrates that the two intestinal β-galactosidases are regulated differently. Our results suggests a complex developmental regulation of human intestinal lactase and that the perinatal increase in lactase activity could be modulated at a post-transcriptional level by hydrocortisone.

Intraluminal and intracellular phases of fat absorption are impaired in essential fatty acid deficiency.

The structure and function of enterocyte membranes are particularly sensitive to the degree of fatty acid saturation. The objective of the present study was to assess intestinal fat transport in essential fatty acid (EFA)-deficient animal models. Both the digestive and absorptive phases leading to the formation and the secretion of triglyceride (TG)-rich lipoproteins were investigated. After an intraduodenal fat infusion, the percentage increase of plasma TG over fasting values was examined over a period of 4 h in two groups of control and EFA-deficient rats. Lower values at 1 and 2 h (P less than 0.05) were observed in EFA-deficient rats, suggesting fat malabsorption. Likewise, postprandial chylomicronemia was diminished. In a separate group of rats, EFA deficiency was associated with reduced TG and chylomicron-TG transport into lymph. Although pancreatic lipase activity did not change (47.1 vs. 46.2 mumol free fatty acids.mg protein-1.h-1), bile flow was decreased over the 8-h period of collection. Concomitantly, a significant decline (nmol.min-1.g liver-1, P less than 0.05) was discernible in the biliary secretory rate of bile salts (14.09 +/- 2.13 vs. 35.09 +/- 3.73), phospholipids (7.01 +/- 0.61 vs. 11.79 +/- 1.65) and cholesterol (0.19 +/- 0.01 vs. 0.83 +/- 0.06). In vitro studies, utilizing everted sacs incubated with mixed micelles, revealed that EFA-deficient jejunal segments of rats incorporated and esterified less [14C]oleic acid (21 and 32%, respectively). Moreover, the synthesis and secretion of TG-rich lipoproteins were found markedly reduced in mouse jejunal explant cultures. We conclude that EFA deficiency modifies both the intraluminal and intracellular phases of fat absorption.

REGULATION OF INTESTINAL SUCRASE EXPRESSION IN SUCKLING MOUSE INTESTINE

Intestinal disaccharidase expression in the developing post-natal animal is under a complex system of regulatory mechanisms involving pre-programmed genetic timing as influenced by circulating humoral factors, paracrine factors, and luminal nutrients. The aim of this study was to examine the relative contribution of circulating glucocorticoids and pre-programmed genetic expression of sucrase and lactase in a suckling mouse between post-natal days 13 and 15. We examined primary jejunal and ileal organ culture explants in serum-free medium at 0-2 hours and 22-24 hours of organ culture, respectively. Animals were studied at days 13, 14, and 15, with an additional group of adrenalectomy animals who underwent adrenalectomy on day 13 with organ culture at day 15. The results of sucrase activity are shown below.In summary, day 14 tissues in organ culture for 24 hours reached the same level of expression in organ culture of scrum-free media as in the intact animal on day 15. In addition, adrenalectomy on day 13 did not abolish the time-dependent expression of sucrase. However, the magnitude of expression in the basal state was diminished. We speculate that normal quantitative expression of intestinal disaccharidase in the 15-day suckling mouse is dependent upon a combination of intrinsic tuning in conjunction with circulating glucocorticoids.

Effect of antifungal agents on the adherence of Candida albicans to murine gastrointestinal mucosal surfaces

The in-vitro adherence of Candida albicans to murine gastrointestinal mucosal surfaces was investigated in the presence of sub-inhibitory concentrations of amphotericin B, ketoconazole and itraconazole. Each antifungal drug showed a significant ability to reduce the adherence of C. albicans to gastric and jejunal mucosa. However, the outcome of an adherence assay was dependent on pH, drug concentration and C. albicans strain. The type of mucosal surface and its cellular arrangement also had an important role to play. Sub-inhibitory concentrations of amphotericin B, ketoconazole and itraconazole significantly reduced the adherence of an azole sensitive strain C. albicans 3436. These effects were influenced by the type of mucosal surface and the concentration of the antifungal drug. Sub-inhibitory concentrations of amphotericin B inhibited the adherence of an azole resistant strain. C. albicans 3310, to stomach and jejunal mucosal surfaces while sub-inhibitory concentrations of ketoconazole only significantly reduced this strain's adherence to gastric mucosal explants. Sub-inhibitory concentrations of itraconazole had little effect. Amphotericin B reduced the adherence of C. albicans 3436 to gastric mucosal explants when assays were performed at physiological pH levels, an effect dependent on concentration. This drug also reduced the adherence of strain 3436 to jejunal mucosal cells and explants at physiological pH levels, though results obtained from assays involving jejunal mucosal explants, incubated at pH 8.0, were concentration dependent.

Suckling Rat Colon Synthesizes and Processes Active Lactase-Phlorizin Hydrolase Immunologically Identical to that from Jejunum

To identify potential tissue-specific characteristics of intestinal glycoprotein synthesis and processing, rat intestinal lactase-phlorizin hydrolase (L-Ph) was studied after pulse-labeling of colonic explants from 5-d-old suckling rats in organ culture and the data compared to similar studies in rat jejunum. Histologic sections of 5-d-old proximal colon showed villus-like structures lined with columnar epithelial cells. Lactase and phlorizin hydrolase activities showed tissue-specific developmental patterns. Using a MAb to small intestinal L-Ph, we were able to immunoprecipitate from colon at different ages a protein that hydrolyzed lactose and phlorizin, and whose activity was not inhibited by p-chloromercuribenzoate. After pulse-labeling for 60 min and chase for 30 min, immunoprecipitated L-Ph from total homogenates of rat colonic explants appeared on fluorography of SDS-PAGE as one band of approximately 205 kD. With increasing time of chase, it took 240 min before the precursor form was converted to the intermediate form (equivalent to the 180-kD form in jejunum) and the mature form (equivalent to the 130-kD form in jejunum), although these conversions in the jejunum were observed within 60 min of chase, and only 30 min of pulse labeling. When compared on SDS-PAGE to immunoprecipitated jejunal L-Ph, the precursor form in the colon had a slightly higher apparent mol wt than the corresponding precursor form found in the endoplasmic reticulum-Golgi fraction of the jejunum. The intermediate as well as the mature L-Ph forms in the colon were also both somewhat higher in apparent molecular weight than the same bands in the microvillus membrane fraction from jejunal explants. Removal of N-linked oligosaccharides from jejunum and colonic forms of L-Ph produced bands on SDS-PAGE with identical mobility, suggesting that the proteins were the same. The data demonstrate that, in neonatal colon, enzymatically active L-Ph undergoes biosynthetic and processing events similar to those in the jejunum. During early life, colonic L-Ph may function in the salvage of lactose not absorbed in the small intestine.

Effect of calcium and magnesium ions on the intestinal absorption of oleic acid in vitro.

The effect of Ca++ and Mg++ upon intestinal absorption of oleic acid was investigated using two in vitro models: rat isolated jejunal loops at 30 degrees C and 37 degrees C and mouse jejunal explants at 37 degrees C. At 30 degrees C or at 37 degrees C, Ca++ significantly increased 14C oleic acid uptake by rat isolated jejunal loops or mouse jejunal explants; at 37 degrees C, Ca++ significantly enhanced lipid exocytosis in rat intestinal loops but not in mouse jejunal explants; in both models, in the presence of Ca++ and at 37 degrees C, Mg++ significantly improved the esterification of oleic acid phospholipids and triacylglycerols, as shown by the increase in triacyglycerol synthesis in rat isolated intestinal loops or by the increase in triacylglycerols recovered from the incubation media of mouse jejunal explants; experiments carried out with rat isolated jejunal loops highlighted the determinant role of temperature in oleic acid absorption processes. The present work shows that the simultaneous presence of Ca++ and Mg++ did not impede oleic acid absorption processes but, on the contrary, enhanced them.

Biosynthesis, glycosylation, and intracellular transport of intestinal lactase-phlorizin hydrolase in rat.

The biosynthesis of rat intestinal lactase-phlorizin hydrolase was studied by pulse-labeling of jejunal explants from 5-day-old suckling rats in organ culture. Explants were either continuously labeled with [35S] methionine for 15, 30, and 60 min or pulse-labeled for 30 min and chased for various periods of time up to 6 h in the presence or absence of protease inhibitors (PI), leupeptin, phenylmethylsulfonyl fluoride, and soybean trypsin inhibitor. Lactase-phlorizin hydrolase was immunoprecipitated from microvillus membrane (MVM) and ER-Golgi fractions with monoclonal antibodies. After pulse-labeling, lactase-phlorizin hydrolase from the ER-Golgi fraction appeared on SDS-PAGE as one band of approximately 220 kDa, regardless of the presence or absence of PI in the culture media. The 220-kDa protein band could also be labeled after incubation with [2-3H]mannose. In the absence of PI, the 220-kDa band appeared in the MVM by 30 min chase, simultaneously with a 180-kDa band, and by 60 min of chase an additional band of 130 kDa was seen. With increasing time of chase, the relative intensity of the 130-kDa band increased, whereas that of the 220-kDa band decreased, suggesting a precursor-product relationship. When PI were added to the medium, the formation of the 180-kDa band was not affected, but the conversion of the 180-kDa protein to the 130-kDa protein was virtually blocked. These findings suggest that lactase-phlorizin hydrolase is initially synthesized as a glycosylated precursor of 220 kDa, which is transported to the MVM. There it undergoes the following two cleavages: first, to the 180-kDa form, which is not prevented by PI used in these experiments, and second, to the 130-kDa form inhibited by PI.

Intestinal apoB synthesis, lipids, and lipoproteins in chylomicron retention disease

Chylomicron retention disease is characterized by fat malabsorption, hypocholesterolemia, normal fasting triglycerides, and marked intestinal steatosis despite the presence of both plasma and intestinal apoprotein B. The defect remains unknown but presumably involves the synthesis or secretion of chylomicrons. The present investigation examines this hypothesis by studying the biosynthesis of chylomicrons in cultured jejunal explants and by defining the quantitative and qualitative abnormalities of plasma lipids and of circulating lipoproteins. Following 2-3 years of a low fat diet supplemented with medium chain triglycerides, six patients with chylomicron retention disease had significantly higher triglyceride (TG) levels coupled with a decrease in both free (FC) and esterified cholesterol (EC) as well as in essential fatty acids and phospholipids (PL) when compared to healthy controls. The low total plasma cholesterol was largely accounted for by low levels of both low density (LDL) and high density lipoprotein (HDL) cholesterol. VLDL and LDL were characterized by a diminished percentage of CE with an increase of TG while HDL contained relatively more FC as well as PL and less CE. The diameter of VLDL was larger whereas those of LDL and HDL were smaller than in normal controls. Jejunal explants, when incubated with [14C]palmitate, were capable of normal biosynthesis of TG, diglycerides, PL, and CE. These lipids, however, except for PL, were retained in the tissue and could not be secreted into the culture medium. Incubation of intestinal biopsies with [3H]leucine and [14C]mannose resulted in normal protein synthesis and reduced glycosylation. The presence of intestinal apoB-48 was confirmed by immunoblot using 2D8 antibodies. These data suggest that the intestinal defect in this disease results from a disorder of the final assembly of chylomicrons or in the mechanism of their exocytosis.

Evidence for a growth-stimulating fraction in the rat proximal intestine after small bowel resection

Small bowel resection results in a compensatory hyperplasia in the small intestine, but the molecular events that lead to the increased cell production are not known. In this study, a heat-stable acidic extract of the mucosa of the proximal intestine of Sprague-Dawley rats taken 96 h after a 50% small bowel resection was capable of stimulating DNA synthesis of mouse jejunal expiants in organ culture. This stimulatory activity was present in the extracts obtained from resected animals after 48 h, 72 h, and 96 h, but was not detectable by 8 days, when presumably a new steady state was established. A significant enhancement of DNA synthesis was observed 96 h after resection when compared with groups of normal and transected animals that were pair-fed with the resected group. This activity was destroyed by protease treatment. Gel filtration experiments showed that the growth-stimulating activity present in the mucosal extract of the 96-h resected animals was due to the presence of two distinct molecules with approximate molecular weights of 4500 and 1500. The extract did not stimulate DNA synthesis in rat peripheral blood lymphocytes, mouse skin fibroblasts, and the colon adenocarcinoma cell line, HCT-8R. Similar extracts taken 96 h after resection from the distal intestine, colon, pancreas, liver, and muscle did not stimulate DNA synthesis of the mouse jejunal explants. These data suggest that the two molecules in the proximal intestine in response to resection could play a role in promoting the observed hyperplasia.

Differential toxicity of RCAII (ricin) on rabbit intestinal epithelium in relation to postnatal maturation.

The purpose of this work was to assess the toxic lectin ricin (RCAII) as a probe for the study of intestinal permeability in the developing small bowel. Jejunal explants from suckling and adult rabbits were exposed to varying dosages of RCAII for 30 min at 25 degrees C and then cultured in toxin-free medium. The RCAII dose required to inhibit protein synthesis during 6 h of culture increased from 0.1 microgram/ml in 4-day-old rabbits to 25 micrograms/ml in weanling rabbits. RCAII cytotoxicity was almost completely blocked by 0.1 M lactulose in all age groups. The kinetics of 125I-RCAII binding to purified microvillus membranes were determined by incubating a fixed concentration of membrane protein (30 micrograms) with increasing concentrations of labeled lectin (2-18 micrograms/ml). Binding attained saturation with adult but not with suckling animal membranes. The latter yielded a curvilinear relationship in Scatchard plots, suggesting either several classes of binding sites or negative cooperativity. RCAII binding was confined to the delipidated fraction of the membranes and decreased by 42% from 6 days old to adult age. The extreme sensitivity of colostral epithelium to RCAII is probably related to the high level of endocytosis exhibited by the immature membrane of suckling rabbits. The development of increasing resistance to the toxin, and associated decrease in binding, might be related to disappearance of saccharide sites in productive surface receptors occurring in the developmental course of intestinal glycosylation.

Effects of epidermal growth factor (EGF) on adult mouse small intestine in vivo and in organ culture

1. 1. Exogenous administration of epidermal growth factor (EGF) has not modified the protein and DNA content, nor several brush border enzymes activities of duodenum, jejunum and ileum of intact and fasted adult mice. 2. 2. Exogenous administration of EGF has not stimulated the DNA synthesis in the three regions of the small intestine of intact adult mice. 3. 3. EGF has a stimulatory effect on DNA systhesis of fasted mice intestine 12 hr after injection. 4. 4. In organ culture, EGF has not altered at any concentration (10, 50, 100, 200, 800 ng/ml), the same parameters in duodenal and jejunal explants taken from animals fasted 24 hr before being killed. 5. 5. These last results suggest that the increase of DNA synthesis observed in vivo was not a direct effect of EGF administration. 6. 6. Finally, the EGF content of serum of adult male mice was measured in fed and fasted mice and in the organ culture media.

277 CHANGES IN SENSITIVITY TO RICINUS COMMUNIS TOXIN (RICIN) OF RABBIT JEJUNUM DURING THE SUCKLING PERIOD

Pinocytosis is the predominant route of penetration of macromolecules across the small bowel epithelium of newborn animals. We have developed a model system to study this process on jejunal mucosal explants using ricin. Ricin is a potent toxin which inhibits protein synthesis by a mechanism involving binding to galactose surface residues, internalization of its enzymatic component and inactivation of the 60 S ribosomal subunit. Protein synthesis (3H-leucine incorporation) in jejunal explants from fetal, suckling and adult rabbits was measured in organ culture following an initial exposure (30 min, 25°) to ricin (1-25 μg). The rate of inhibition of protein synthesis was age dependent (Fig). High in the fetus, it increased further to a maximum at day 6 postnatally, then decreased rapidly in suckling animals to adult levels at weaning. The high sensitivity to ricin during the colostral period may be related to the following factors currently under study: 1) colostral-milk components stimulating a high rate of endocytosis, 2) transitional maturation changes in membrane properties and 3) changes in sensitivity at the target ribosomal site. The model presented is useful to study modifiers and events influencing mucosal permeability in the developing small intestine.

560 EFFECT OF RICINUS COMMUNIS TOXIN (RICIN) ON RABBIT INTESTINAL EPITHELIUM: A MODEL OF PROTEIN-INDUCED MUCOSAL DAMAGE

Ricin inhibits protein synthesis in eukaryotic cells following binding to galactose membrane receptors and cytoplasmic internalization of its A subunit. Similar but less well defined mechanisms are probably involved in small bowel toxicity by certain food and bacterial proteins. We have therefore used ricin to develop an in vitro model of protein-induced small bowel epithelial damage using organ culture methodology. Rabbit jejunal mucosal explants were exposed (30 min, 25°) to varying doses of ricin (0.1-50 μg) and then cultured in ricin-free medium for 2 to 24 hours at 37°. Inhibition of protein synthesis (14C-leucine incorporation) occurred as a function of ricin dose and culture time. A t50 plot (1/ricin/t50) revealed a lag time of about 30 minutes for infinite ricin dose. Light and electron microscopy showed a progression of damage in epithelium which correlated to ricin dose and level of inhibition of protein synthesis. The effect of ricin was blocked by lactose and lactulose (100 mM) added during the ricin exposure period. The dose and time relationships of the effect of ricin on intestinal epithelium in culture suggest membrane responses and down regulation processes identical to those of endocytotic cells. This approach is therefore useful for investigations of the membrane and cellular mechanisms associated with protein-induced enterocyte toxicity.

49: Glycoprotein synthesis by human small intestine in organ culture

Glycoprotein synthesis was measured by the incorporation of 14-C-glucosamine into total acid precipitable protein of jejunal explants cultured in vitro. The rate of glucosamine incorporation remained constant up to 48 hours. During this time a continuous release of glycoprotein into the culture medium was observed. Pulse-chase experiments indicated that the mean half life of overall labelled tissue glycoprotein was about 20 hours. SDS-polyacrylamide-gel-electrophoresis of the isolated brush border membrane showed that all major protein bands of high molecular weight had incorporated 14-C-glucosamine. These proteins were identified as maltase-glucoamylase, lactase, sucrase-isomaltase, enterokinase and alkaline phosphatase. Specific enzyme activity could not be assigned to the two remaining labelled bands. After 5 hours of culture lactase showed the highest glucosamin incorporation. These results indicate that the organ culture method is suitable for studying the biosynthesis, of human intestinal brush border enzymes in biopsies from patients with intestinal malabsorption syndromes.