The effect of cytokines on intestinal sugar absorption during sepsis in rabbits.

The endotoxin that triggers an immune response to Gram-negative bacterial infection namely lipopolysaccharide (LPS), is also associated with gastrointestinal abnormalities and induces the release of proinflammatory cytokines such as IL-1 and TNF-α. The main aim of this study was to determine the effect of cytokine release on intestinal D-fructose absorption in LPS-treated rabbits in order to provide information that could be used to understand their septic status. The results obtained, using whole tissue and brush border membrane vesicles from rabbit jejunum, showed that LPS, TNF-α and IL-1ß inhibit d-fructose absorption across the jejunum. The effect of LPS is completely reversed by a TNF-α antagonist and partially by a specific IL-1 receptor antagonist (IL-1ra) and disappears completely in the presence of both these cytokine antagonists. Similarly, the effects of TNF-α and LPS were not totally blocked by IL-1ra, whereas the effect of IL-1ß disappeared completely in the presence of a TNF antagonist. In summary, these results show that TNF-α and IL-1ß could act synergistically on sugar absorption in rabbit with LPS-induced sepsis. In addition, the effects of IL-1ß depend on, or are related to TNF-α production since this effect returns to basal (control) levels in the presence of a TNF-α antagonist.

Intestinal D-galactose transport in an endotoxemia model in the rabbit.

Lipopolysaccharide (LPS) is an endotoxin causing sepsis. Studies from our laboratory revealed impaired intestinal absorption of L-leucine and D-fructose in LPS-treated rabbits. The aim of this study was to examine intestinal D-galactose transport following intravenous administration of LPS in the rabbit and to identify the cellular mechanisms driving this process. Endotoxin treatment diminished the buildup of D-galactose in intestinal tissue, the mucosal to serosal transepithelial flux of the sugar and its uptake by brush border membrane vesicles (BBMVs). Intracellular signaling pathways associated with protein kinase C (PKC), protein kinase A (PKA), p38 mitogen-activated protein kinase (p38MAPK), Jun N-terminal kinase (JNK), MAPK/extracellular signal-regulated kinases 1 and 2 (MEK1/2) and proteasome were found to be involved in this reduction in sugar uptake. Na(+)/glucose cotransporter 1 (SGLT1) protein levels in BBMVs were lower for LPS-treated animals than control animals. These findings indicate that LPS inhibits the intestinal absorption of D-galactose via a complex cellular mechanism that could involve posttranscriptional regulation of the SGLT1 transporter.

Inhibitory effect of TNF-alpha on the intestinal absorption of galactose.

Sepsis is a systemic response to infection in which toxins, such as bacterial lipopolysaccharide (LPS), stimulate the production of inflammatory mediators like the cytokine tumor necrosis factor alpha (TNF-alpha). Previous studies from our laboratory have revealed that LPS inhibits the intestinal absorption of L-leucine and D-fructose in rabbit when it was intravenously administered, and that TNF-alpha seems to mediate this effect on amino acid absorption. To extend this work, the present study was designed to evaluate the possible effect of TNF-alpha on D-galactose intestinal absorption, identify the intracellular mechanisms involved and establish whether this cytokine mediates possible LPS effects. Our findings indicate that TNF-alpha decreases D-galactose absorption both in rabbit intestinal tissue preparations and brush-border membrane vesicles. Western blot analysis revealed reduced amounts of the Na+/glucose cotransporter (SGLT1) protein in the plasma membrane attributable to the cytokine. On the contrary, TNF-alpha increased SGLT1 mRNA levels. Specific inhibitors of the secondary messengers PKC, PKA, the MAP kinases p38 MAP, JNK, MEK1/2 as well as the proteasome, diminished the TNF-alpha-evoked inhibitory effect. LPS inhibition of the uptake of the sugar was blocked by a TNF-alpha antagonist. In conclusion, TNF-alpha inhibits D-galactose intestinal absorption by decreasing the number of SGLT1 molecules at the enterocyte plasma membrane through a mechanism in which several protein-like kinases are involved.

The effect of tumor necrosis factor-alpha on D-fructose intestinal transport in rabbits.

Tumor necrosis factor-alpha (TNF-alpha) is an important immunoregulatory cytokine involved in septic responses during bacterial infection. The aim of this study was to examine the effect of TNF-alpha on the transport of D-fructose across rabbit jejunum. A sepsis condition was evoked by intravenous administration of this cytokine and hematological and plasma parameters were analyzed and body temperature was recorded. D-Fructose transport was assayed in rabbit jejunum. Sugar absorption in TNF-alpha treated rabbits was lower than in control animals. TNF-alpha decreased both the mucosal-to-serosal transepithelial flux and the transport across brush border membrane vesicles of D-fructose. The number of D-fructose transporters (GLUT5) was analyzed by Western blot in an attempt to explain this inhibition. TNF-alpha treated animals had lower levels of GLUT5, indicating a reduction in the expression of GLUT5 protein and therefore in transport capacity. The inhibition could also be related with the secretagogue effect of TNF-alpha on the gut since the intracellular tissue water was affected and the absence of chloride ion in the incubation medium partly removed the cytokine inhibition on sugar intestinal transport in treated rabbits. Finally, in terms of possible mediators involved in the TNF-alpha effect, nitric oxide and prostaglandins appeared to play a role in the inhibition of D-fructose intestinal uptake.

Effect of lipopolysaccharide on D-fructose transport across rabbit jejunum.

OBJECTIVE: To investigate alterations in the transport of D-fructose across the rabbit jejunum when the gut is exposed in vitro to lipopolysaccharide (LPS), an endotoxin causative agent of sepsis. MATERIALS AND METHODS: D-fructose intestinal transport was assesed employing three techniques: sugar uptake measurements in rings of everted jejunum (micromol/D-fructose/ml cell water), transepithelial flux measurements in Ussing-type chambers (micromol D-fructose/cm2/h) and transport assays in preparation of brush border membrane vesicles (pmoles D-fructose/mg protein). Samples were taken from the bathing solution and from the extracts of the tissue for radioactivity counting. RESULTS: Adding LPS (3 microg/ml) to tissue decreased the uptake and mucosal to serosal flux of 5 mM D-fructose across the enterocyte. LPS did not modify sugar uptake across brush border membrane vesicles. The inhibitory effect of LPS was suppressed by W-13 (5 x 10(-6) M), a Ca-calmodulin antagonist, and staurosporine (10(-7) and 10(-6) M) and GF-109203X (10(-6) M) a nonselective and selective protein kinase C (PKC) inhibitor respectively. Tumor Necrosis Factor (TNF-alpha), an immunoregulatory cytokine involved in septic responses occurring during bacterial infection at concentrations 3 x 10(-4) to 3 microg/ml, did not affect the sugar transport. CONCLUSIONS: LPS can inhibit the intestinal uptake of D-fructose across the rabbit jejunum in vitro by intracellular processes related to PKC and calmodulin protein.

Tumor necrosis factor-alpha mediates inhibitory effect of lipopolysaccharide on L-leucine intestinal uptake.

Tumor necrosis factor-alpha (TNF-alpha) has been proposed as an early proximal mediator of many metabolic and physiologic responses during septic shock. We have previously shown that direct addition to tissue (local effect) or intravenous administration (systemic effect) of lipopolysaccharide (LPS) reduces L-leucine absorption across rabbit jejunum. In the present study, we investigated whether the inhibitory effect of LPS on L-leucine intestinal absorption in rabbit is related to TNF-alpha. The results shown that the addition of TNF-alpha to tissue does not produce any effect on L-leucine uptake by the enterocyte. When TNF-alpha was inoculated by intravenous administration, a strong inhibition on the L-leucine uptake (about 40%), mediated by a secretagogue effect on water and Cl-ions was induced. We also found that the LPS intestinal effect induced by intravenous administration, was blocked by a TNF-alpha antagonist, indicating that TNF-alpha is a mediator of the LPS systemic effect on L-leucine intestinal uptake inhibition. The study of possible mediators involved in the TNF-alpha effect showed that nitric oxide and prostaglandins are implicated in the L-leucine intestinal uptake.

Cellular mechanism underlying LPS-induced inhibition of in vitro L-leucine transport across rabbit jejunum.

Lipopolysaccharide (LPS) is a known causative agent of sepsis. In previous studies, we have shown that it reduces L-leucine mediated transport across the rabbit jejunum by about 30%. In this study, the mechanism(s) of LPS inhibition on amino acid transport were analysed in detail. LPS did not inhibit L-leucine transport across brush border membrane vesicles, suggesting the need for an intracellular step. The inhibitory effect of LPS was not altered by the addition of protein kinase A (PKA) inhibitor (IP(20), 10(-7) M) or an analog of cAMP (DB-cAMP, 3 x 10(-4) M), indicating that the PKA signal transduction pathway was not involved in the LPS effect. However, the inhibitory effect of LPS was suppressed by trifluoroperazine (10(-7) M), a Ca(2+)/calmodulin inhibitor and staurosporine (10(-7) M), an protein kinase C (PKC) inhibitor. Likewise, LPS inhibition disappeared in media without calcium. These results suggest that LPS could inhibit the intestinal uptake of L-leucine across the small intestine in vitro by intracellular processes related to calcium, involving PKC and calmodulin protein.

Effect of lipopolysaccharide on small intestinal L-leucine transport in rabbit.

In the present study, we have investigated whether the lipopolysaccharide (LPS) endotoxin from Escherichia coli is able to alter the jejunal transport of L-leucine when the tissue is exposed to endotoxin. The results have shown that the LPS at 3 x 10(-5) microg/ml decreases the uptake of L-leucine into the enterocyte, as well as the mucosal to serosal flux of L-leucine. The secretagogue effect of LPS on the gut did not affect the inhibitory effect of LPS on the intestinal absorption of the amino acid. The endotoxin did not modify amino acid diffusion across the intestinal epithelium. However, from the mediated transport, only the Na+-dependent transport system was affected by LPS with a diminution of the transporter affinity (the apparent Km was increased). In addition, we found a reduction of the Na+, K+-ATPase activity, which could explain the L-leucine Na+-dependent transport inhibition.

The administration of lipopolysaccharide, in vivo, induces alteration in L-leucine intestinal absorption.

The objective of the present study was to determine the alterations in L-leucine intestinal uptake by intravenous administration of Lipopolysaccharide (LPS), which is a constituent of gram negative bacterial, causative agent of sepsis. The amino acid absorption in LPS treated rabbits was reduced compared to the control animals. The LPS effect on the amino acid uptake was due to an inhibition of the Na+-dependent system of transport, through both reduction of the apparent capacity transport (Vmax) and diminution of the Na+/K-ATPase activity. The results have also shown that the LPS decreases the mucosal to serosal transepithelial flux and the transport across brush border membrane vesicles of L-leucine. The study of possible intracellular mechanisms implicated in the LPS effect, showed that the second messengers calcium, protein kinase C and c-AMP did not play any role in this effect. However, the absence of ion chloride in the incubation medium removes the LPS inhibition and the intracellular tissue water was affected by the LPS treatment. Therefore, the inhibition in the L-leucine intestinal absorption, by intravenous administration of LPS, could be mainly produced by the secretagogue action of this endotoxin on the gut.

Study of serotonin interactions with brush border membrane of rabbit jejunum enterocytes.

Recent studies have demonstrated that serotonin (5-hydroxytryptamine, 5-HT) may interact with either specific receptors or with a specific transporter that takes up 5-HT in the gastrointestinal tract. The purpose of the present work was to study the 5-HT interactions with brush border membrane from rabbit jejunum enterocytes. The results obtained showed that 5-HT did not seem to be transported by any specific system of transport in brush border membrane vesicles. Nevertheless, [3H]5-HT seemed to bind specifically to this membrane. The kinetic analysis indicated a saturable and dissociable specific binding with a dissociation constant K(D)=14x10(-9) M. The saturation studies with [3H]5-HT indicated the presence of one specific site in the brush border membrane. The results of displacement of [3H]5-HT specific binding from the brush border membrane showed that both unlabelled 5-HT and unlabelled GR113080 ([1-[(2-methyl sulphonyl) amino] ethyl-4-piperidinyl] methyl-1-methyl-1H-indole-3-carboxylate), a specific competitive antagonist of 5-HT(4) receptors, inhibited the specific binding of [3H]5-HT to this membrane.

Effects of serotonin on the physiology of the rabbit small intestine.

Serotonin has been shown to alter the intestinal transport of ions and intestinal motility. These effects may interfere with each other, modulating the whole physiology of the intestine. We have previously shown that serotonin also alters the transport of nutrients. Thus, the aims of the present work were to determine the possible interference between the secretagogue effect of serotonin and the mechanism by which serotonin inhibits the absorption of nutrients, and to study the effect of serotonin on the digestive activity of nutrients of the brush border membrane jejunum enterocyte in the rabbit. The results show that the secretagogue effect of serotonin neither affects the inhibitory effect of serotonin on the intestinal absorption of the nutrients, nor affects the activity of Na+/K+-ATPase. The activity of sucrase and aminopeptidase N was also not affected by serotonin in the rabbit jejunum. Finally, we also studied different parameters of the motility in the rabbit small intestine. Serotonin seemed to stimulate the motility of the rabbit small intestine by increasing integrated mechanical activity and tone of muscle fibers in duodenum, jejunum, and ileum. In conclusion, serotonin might alter or modulate the whole intestinal physiology.

5-HT receptor subtypes involved in the serotonin-induced inhibition of L-leucine absorption in rabbit jejunum.

The aim of the present study was to determine the 5-HT receptor subtypes involved in the serotonin-induced inhibition of L-leucine absorption across rabbit jejunum in vitro. A number of agonists and antagonists were used to characterize the receptors through which serotonin inhibits this absorption. The results show that 2.5x10(-6) M 5-HT inhibits the amino acid absorption by about 20%. The 5-HT receptor agonists, alpha-methyl-5-HT (5-HT2), 2-methyl-5-HT (5-HT3) and zacopride (5-HT4) at concentrations 2.5x10(-6) and 2.5x10(-5) M produced 10-30% inhibition on L-leucine intestinal absorption. 5-carboxyamidotryptamine (5-HT1) did not produce any inhibition. The 5-HT antagonists, GR 113808A (5-HT4) at 2.5x10(-6) M and ritanserin (5-HT2) and ondansetron (5-HT3) at 2.5x10(-5) M completely blocked the effect of 5-HT. However, methiothepin (5-HT1) did not produce any effect on serotonin action in the intestinal absorption of amino acid. It can be concluded that 5-HT2, 5-HT3 and 5-HT4 receptors could mediate inhibition of L-leucine absorption across rabbit jejunum.

Inhibition of L-threonine intestinal absorption in rabbits by cadmium.

Cadmium compounds are widely spread in the environment. Animal exposure to cadmium compounds occurs mainly through foods or drinks contaminated by this metal. Cadmium has been shown to produce several negative effects on the gastrointestinal tract such as inhibition on sugars and amino acids absorption. The aim of the present work was to study the inhibitory characteristics of cadmium on L-threonine intestinal absorption in rabbits in order to understand about this malabsorption of nutrients. Our results show that L-threonine tissue accumulation as well as mucosal to serosal transepithelial fluxes are decreased in a dose-dependent manner in rabbit jejunum. Amino acid diffusion across the intestinal epithelium was not affected by cadmium. A noncompetitive mechanism and a partial reversion by dithioerythritol (thiol groups protector) is described for this inhibition.

Effect of zinc on aminopeptidase N activity and L-threonine transport in rabbit jejunum.

Zinc is a nutritionally essential trace element required for many biological functions to be successfully carried out. The aim of the present work was to study the influence of zinc on the intestinal absorption of L-threonine and on the aminopeptidase N activity in rabbit jejunum, after in vitro addition and/or oral administration of ZnCl2 in drinking water. Results obtained show that zinc decreases L-threonine absorption in the jejunal tissue. This effect would appear to be owing to an action mainly located in active amino acid transport, because zinc does not seem to modify the amino acid diffusion across the intestinal epithelium, of the mucosal border of the intestinal epithelium. Zinc has also been shown to inhibit the (Na(+)-K+)-ATPase activity of the enterocyte, which might explain the inhibition of the L-threonine Na(+)-dependent transport. Nevertheless, a direct action of the zinc on carriers of active transport cannot be rejected. However, zinc did not significantly modify the aminopeptidase N activity in rabbit jejunum.

Serotonin-induced changes in L-leucine transport across rabbit jejunum.

The aim of the present study has been to determine the effect of serotonin (5-HT) on the absorption of L-leucine across the rabbit jejunum. The results show that serotonin significantly diminishes the uptake and steady-state tissue accumulation and the mucosal to serosal flux of L-leucine. This effect does not change with previous intestinal exposure of the mucosa to the 5-HT. Serotonin does not seem to modify amino acid simple diffusion across the intestinal epithelium. The effect on the amino acid uptake is due to an inhibition of the Na(+)-dependent system of transport, mainly through a reduction of the apparent Vmax. Moreover, this hormone, added to the incubation solution, does not affect the L-leucine uptake across brush border membrane vesicles. In presence of trifluoroperazine, TMB-8 and staurosporine, the serotonin effect disappears. These results suggest an effect mediated by intracellular processes related to protein kinase C which inhibit the intestinal absorption of L-leucine.

Sugar-dependent expression of the fructose transporter GLUT5 in Caco-2 cells.

The effect of glucose and fructose and fetal bovine serum on the expression of the fructose transporter GLUT5 was studied in clone PD7 of the human colon cancer cell line Caco-2, which has been characterized previously [Chantret, Rodoloswe, Barbat et al. (1994) J. Cell Sci. 107, 213-225; Mahraoui, Rodolosse, Barbat et al. (1994) Biochem. J. 298, 629-633]. Culture of the cells in dialysed serum and hexose-free media, down-regulated the expression of GLUT5, which was below detection within 3-4 days. This effect was reversed by fructose and glucose feeding of the cells. Fructose feeding yielded a 3-fold higher abundance of GLUT5 protein and mRNA as compared with that expressed in glucose-fed cells. Cells fed normal serum exhibited an inverse hierarchy of expression, with glucose being a better inducer than fructose for the expression of GLUT5. The GLUT5 mRNA and protein abundances obtained in fructose-fed cells did not depend on the type of serum. A linear relationship between cyclic AMP (cAMP) levels and GLUT5 mRNA abundance was found in cells fed dialysed serum, whereas in cells fed normal serum, mRNA abundances were not correlated to cAMP levels. These results indicate that glucose and fructose, together with serum-related factors and cAMP, have combined effects on the expression of GLUT5 in Caco-2 cells.

Study of interaction between calcium and zinc on D-galactose intestinal transport.

Zinc is an essential trace element necessary to life. This metal may exert some of its physiological effects by acting directly on cellular membranes, either by altering permeability or by modulating the activity of membrane-bound enzymes. On the other hand, calcium is an essential element in a wide variety of cellular activities. The aim of the present work was to study a possible interaction between zinc and calcium on intestinal transport of D-galactose in jejunum of rabbit in vitro. In media with Ca2+, when ZnCl2 was present at 0.5 or 1 mM, zinc was found to reduce the D-galactose absorption significantly. In Ca(2+)-free media, where CaCl2 was omitted and replaced isotonically with choline chloride, the sugar transport was not modified by zinc. Verapamil at 10(-6) M (blocking mainly Ca2+ transport) did not modify the inhibitory effect of zinc on D-galactose transport. When 10(-6) M of A 23187 (Ca(2+)-specific ionophore) was added with/without Ca2+ to the media, ZnCl2 produced no change in sugar transport. These results could suggest a possible interaction of calcium and zinc for the same chemical groups of membrane, which could affect the intestinal absorption of sugars.

Interaction between calcium and zinc on L-threonine absorption in rabbit jejunum.

The essential minerals calcium and zinc serve unique functions in higher organisms, and it is well recognized that homeostatic mechanisms are involved in regulating their metabolism. However, it has been reported that zinc, at higher concentrations (1 mM), inhibits intestinal absorption of sugars and amino acids. The aim of the present work was to determine whether the inhibitory effect on L-threonine absorption across the rabbit jejunum could be modified by calcium. In media with Ca2+, zinc significantly reduced L-threonine absorption. In Ca(2+)-free media, where calcium chloride was omitted and replaced isotonically with choline chloride, the amino acid transport was not modified by zinc, but when calcium chloride was replaced isotonically with magnesium chloride, the inhibition was observed. Verapamil (blocking mainly Ca2+ transport) did not modify the inhibitory effect of zinc on L-threonine transport. When A23187 (Ca(2+)-specific ionophore) was added in media with and without Ca2+, zinc produced no change in L-threonine transport. These results suggest that calcium and zinc could have an affinity with the same chemical groups of the enterocyte membrane, which would be related to the intestinal absorption of amino acids.

Calcium-cadmium interaction on L-threonine intestinal transport.

Cadmium is a highly toxic metal that can damage a number of organs including the gastrointestinal tract. It has been shown that cadmium partially reduces L-threonine intestinal absorption probably by binding to membrane proteins which pertain to active transport systems or are functionally related to them. Calcium, however, is an essential element in a wide variety of cellular activities. The aim of the present work was to study whether the inhibitory cadmium effect on L-threonine absorption across rabbit jejunum could be modified by calcium. In media with Ca2+, cadmium significantly reduces the L-threonine absorption. In Ca(2+)-free media, where calcium chloride was omitted and replaced isotonically with choline chloride, this amino acid transport was not modified by cadmium but it was inhibited when calcium chloride was replaced isotonically with magnesium chloride. Verapamil (blocking mainly Ca2+ transport) did not modify the inhibitory effect of cadmium on L-threonine transport. When A 23187 (Ca2+ specific ionophore) was added in media with/without Ca2+, cadmium produced no change on L-threonine transport. These results suggest that calcium and cadmium could have an affinity for the same chemical groups on the enterocyte membrane. This property could affect the intestinal absorption of amino acids.

Effect of motilin on the L-leucine transport in rabbit jejunum.

Motilin is a gastrointestinal peptide that stimulates the gastrointestinal motility in several species. The aim of the present work has been to determine the effect of motilin on the L-leucine absorption in rabbit jejunum. The results show that motilin inhibits the L-leucine Na(+)-dependent system of transport located in the mucosal border mainly by diminishing the apparent Vmax. Motilin did not directly affect the Na(+)-dependent system of transport, but it seems to act across the protein kinase C (PKC). These results suggest that motilin may act as a regulatory hormone of the intestinal absorption of nutrients.