Nippostrongylus brasiliensis: malabsorption in experimentally infected rats

Glucose absorption and net small intestinal water movement were examined in rats infected with Nippostrongylus brasiliensis at Days 4, 6, 9, 13, and 19 after inoculation. Rats were infected with 4 X 10(3) N. brasiliensis third stage larvae. The entire small intestine was divided into three segments and each segment perfused simultaneously in vivo with Krebs-Ringer phosphate buffer containing 80 mM glucose, 6 X 10(5) dpm/ml [3H]glucose, and 6.2 X 10(3) dpm/ml [14C]polyethylene glycol. Rats perfused on Days 6, 9, 13, and 19 after inoculation showed a significant (P less than 0.05) decrease in glucose absorption rates from all three segments of the small intestine when compared to uninfected controls. In the three segments of uninfected rat small intestine and those perfused on Days 4, 13, and 19 after inoculation, net absorption of water occurred. However, in the proximal and distal segments perfused on Day 6 and the proximal segment perfused on Day 9, net water movement into the lumen occurred. This is the first report of depressed glucose absorption along the entire length of the small intestine during nippostrongylosis and contradicts previous reports of unaltered net glucose absorption in response to this parasite.     

Kinetic parameters of maltose hydrolysis and glucose intake in the rat small intestine in a chronic experiment

"True" (corrected for the influence of the pre-epithelial layer) kinetic constants of maltose hydrolysis (Km and Vmax) and Glucose active transport (Kt and Jmax) in the isolated loop of the rat small intestine in chronic experiments were determined using a new mathematical approach. The Km (4.260.25 mM) does not differ from that, obtained in in vitro experiments on the homogenates of mucous membrane taken from the same intestinal loops, and the Vmax (0.72 +/- 0.07 mol/(min.cm)) is 1.7 times lower than that in in vitro experiments. The Kt and Jmax values are 3.18 +/- 0.68 mM and 0.73 +/- 0.07 mol/(min.cm), resp. The estimated values of Km, Kt and Vmax are in accordance with the corresponding published data, whereas the Jmax is several times higher than the value generally believed on the basis of acute experiments in vivo. A high level of glucose absorption in the small intestine of unanesthetized animals is achieved mainly due to a high permeability of the pre-epithelial layer and a high capacity of the active transport as a major mechanism of glucose absorption in the small intestine under normal conditions.     

Inhibition of intestinal sugar transport by phenformin.

The effect of phenformin on the absorption of D-glucose and D-galactose by hamster and rat intestine, was studied. Phenformin did not affect D-glucose absorption by rat intestine, but it inhibited at 10(-3) to 10(-2) M the absorption of D-glucose and D-galactose by hamster intestine. The inhibition was higher when D-glucose was tested. Phenformin also inhibited active accumulation of these sugars by rings of hamster small intestine, in vitro; this effect was greater when D-glucose was utilized. The drug inhibits the oxygen uptake in the tissue in the absence or in the presence of added substrate. Phenformin, as previously suggested, does not seem to act as a specific inhibitor on D-glucose transport, but most likely by its inhibitory effect on mitochondrial respiration.     

Physiological regulation of transepithelial impedance in the intestinal mucosa of rats and hamsters

Isolated intestinal segments from rats or hamsters were recirculated with balanced salt solutions containing fluorocarbon emulsion to provide 6 vpc oxygen. The lumen contained an axial Ag-AgCl electrode, and the serosal surface was surrounded by a cylindrical shell of Ag-AgCl. Transmural impedances were measured at frequencies from 0.01-30 kHz before and after removal of the mucosal epithelium. The resistance of intercellular junctions, RJ, the distributed resistance of the lateral spaces, RL, and the distributed membrane capacitance, CM, were computed from the relations between frequency and impedance. Activation of Na-coupled solute transport by addition of glucose, 3-0-methyl glucose, alanine or leucine caused two- to threefold decreases of transepithelial impedance. Typical changes induced by glucose in hamster small intestine were RJ 30----13 omega, RL 23----10 omega, and CM 8----20 microF (per cm length of segment). Half maximal response occurred at a glucose concentration of 2-3 mM. The area per unit path length of the junctions (Ap/delta chi = specific resistance divided by RJ) in glucose activated epithelium was 3.7 cm in hamster midgut and 6.8 cm in rat. These values are close to the 4.3 cm estimated independently from coefficients of solvent drag and hydrodynamic conductance in glucose-activated rat intestine in vivo. The transepithelial impedance response to Na-coupled solute transport was reversibly dependent upon oxygen tension. It is proposed that activation of Na-coupled solute transport triggers contraction of circumferential actomyosin fibers in the terminal web of the microvillar cytoskeletal system, thereby pulling apart junctions and allowing paracellular absorption of nutrients by solvent drag as described in the previous accompanying paper. Anatomical evidence in support of this hypothesis is presented in the following second accompanying paper.     

Kinetic analysis of glycine and glycylglycine absorption in rat small intestine in chronic experiment

Kinetics of glycylglycine hydrolysis and absorption as well as that of free glycine absorption in isolated loop of the small intestine was studied in chronic experiments in two groups of rats. In the 1st group (n = 5), the isolated loop daily received for 1 or two hours a glucose load (25 mM), whereas in the 2nd group (n = 4)--a glutamic acid load (25 mM). The "true" values (i.e. corrected for the influence of the pre-epithelial layer) of the Michaelis constant for dipeptide transport were lower than those for the free glycine transport: 16 +/- 1.8 versus 36.3 +/- 3.7 mM (in the 1st group) and 15.9 +/- 2.2 versus 34.0 +/- 3.7 mM (in the 2nd group), whereas values of the maximal rate of active transport as calculated per 1 cm of the intestine length were, on the contrary, higher: 0.64 +/- 0.06 versus 0.42 +/- 0.10 mumol/(min.cm) 1st group and 0.86 +/- 0.13 versus 0.56 +/- 0.04 mumol/(min.cm) in the in the 2nd group. It has been shown that, under these conditions, regarded as the most physiological, over 90% of glycylglycine is absorbed via the peptide transport system. Only a small part of this dipeptide amount (less than 10%) splits during membrane hydrolysis with subsequent absorption of the derived glycine. It has also been found that glutamic acid solution as a regular substrate load is more effective (as compared with the glucose solution) in retarding the atrophic changes occurring in the isolated intestine loop and in preserving its structural and functional parameters on a higher level.     

Intestinal absorption of copper: effect of sodium

The mechanisms of copper (Cu) absorption from the small intestinal lumen are poorly understood. In this study we investigated the role of sodium (Na) during the removal of Cu from the lumen of jejunal and ileal segments, using an in situ perfusion procedure in the anesthetized rat. Intestinal absorption of Cu from a 31 microM solution was highest in the presence of an isotonic concentration of NaCl, as compared to solutions containing either glycerol (GRL) or N-methyl-D-glucamine (NMG) as osmotic agents. In the jejunum, mean +/- SEM Cu absorption rates in the presence of the following solutes were: with NaCl, 57.5 +/- 10.5 pmole/min X cm; with GRL, 13.3 +/- 14.7 (P less than 0.05); with NMG, 18.4 +/- 10.1 (P less than 0.05). In the ileum, copper absorption in the presence of NaCl was 64.4 +/- 9.6; with GRL, 24.3 +/- 10.1 (P less than 0.01); with NMG, 15.8 +/- 3.7 (P less than 0.001). Kinetic analysis of the carrier-mediated component of Cu absorption in rat jejunum yielded a Vmax = 47.5 pmole/min X cm and an apparent Kt = 21 microM. The diffusion coefficient was calculated to be 1.4 X 10(-5) cm2/sec. The absorption of Cu was independent of net water absorption, which was highest in the presence of GRL and abolished and reversed into secretion by NMG. The data obtained are indicative of a significant role of Na in the small intestinal transport of Cu, in vivo, although not directly related to unidirectional water fluxes. The cation specificity of Na in this process remains to be elucidated, although the results support earlier studies which postulated that mediated transport may constitute a major component of Cu absorption in the mammalian small intestine.     

Structural and functional analysis of glucose adsorption at high maltose concentrations in the rat small intestine in vivo

To elucidate the mechanism of glucose absorption at high substrate concentrations, we studied structural and ultrastructural peculiarities of enterocytes arranged at different levels along the intestinal villus. The preparations were obtained from an isolated segment of the rat small intestine after its perfusion with maltose solutions with both low (25 mM) and high (100 mM) concentrations, respectively. Under conditions of chronic experiment at high substrate concentration, an enlargement of intercellular clefts, indicating glucose absorption, occurred in deeper areas of the villus. Besides, also in chronic experiment, we studied kinetics of maltose hydrolysis and derived glucose absorption in the isolated segment of the rat small intestine after its perfusion with maltose at superhigh (up to 200 mM) initial concentrations. Based on these data, a conclusion is made that active transport is the main mechanism of absorption of glucose derived from maltose hydrolysis, operating both at low disaccharide concentrations, and in the range of its superhigh (up to 200 mM) concentrations.     

Effects of malnutrition in utero and during lactation on various parameters of the small intestine in rats

Short and long term effects of malnutrition on the small intestine, applied to the rat in uterus and lactation, have been studied. Malnutrition was induced by feeding the pregnant rats on 14 g daily during pregnancy and 21 g during lactation. In the pups (0, 15, 30, 90 and 150 days old), body weight and wet and dry weight and length of small intestine were measured. At 2.5-3 months of age, food transformation efficiency was studied, at 3 and 5 months of age in vivo intestinal absorption of D-glucose (11 mM) was measured. The results indicate a significant decrease in intestinal morphometric parameters in malnourished animals from birth to the age of 5 months. At the age of 3 months both food transformation efficiency and in vivo absorption of glucose were significantly higher in early undernourished animals, whereas at 5 months, glucose absorption was significantly higher in control. It can thus be concluded that early malnutrition altered the small intestine development and functionality and that total recovery did not occur after 4 months on a normal diet.     

The stimulation of glucose absorption and metabolism in rat jejunum by bradykinin: dependence on the composition of commercial diets

Rats were maintained on one of two standard commercial chow diets, Oxoid modified 41B or Bantin & Kingman rat and mouse diet, which differ in that linoleic acid comprises 27% and 44% of their total fatty acid content, respectively: the effects of bradykinin on the absorption, transmural transport and metabolism of glucose (5 mM) were then measured by the perfusion of isolated jejunal loops in vitro. With intestine from rats fed the Oxoid diet, bradykinin (100 nM in the serosal medium) caused significant increases in the rates of glucose absorption (34%, P less than 0.01) and lactate production (69%, P less than 0.01). These bradykinin-stimulated rates were the same, within experimental error, as those observed in the absence of bradykinin with intestine taken from rats fed the Bantin & Kingman diet and on which bradykinin had no effect. It is concluded that feeding rats with different commercial brands of apparently similar laboratory chow diets may result in significantly altered steady-states of glucose homeostasis in rat small intestine and in quite different sensitivities of glucose homeostasis to bradykinin. The possibility is considered that the differences in absorption might result in part from differences in the proportion of linoleic acid, which is known to enhance glucose absorption.     

SMIT2 mediates all myo-inositol uptake in apical membranes of rat small intestine

This study presents the characterization of myo-inositol (MI) uptake in rat intestine as evaluated by use of purified membrane preparations. Three secondary active MI cotransporters have been identified; two are Na(+) coupled (SMIT1 and SMIT2) and one is H(+) coupled (HMIT). Through inhibition studies using selective substrates such as d-chiro-inositol (DCI, specific for SMIT2) and l-fucose (specific for SMIT1), we show that SMIT2 is exclusively responsible for apical MI transport in rat intestine; rabbit intestine appears to lack apical transport of MI. Other sugar transport systems known to be present in apical membranes, such as SGLT1 or GLUT5, lacked any significant contribution to MI uptake. Functional analysis of rat SMIT2 activity, via electrophysiological studies in Xenopus oocytes, demonstrated similarities to the activities of SMIT2 from other species (rabbit and human) displaying high affinities for MI (0.150 +/- 0.040 mM), DCI (0.31 +/- 0.06 mM), and phlorizin (Pz; 0.016 +/- 0.007 mM); low affinity for glucose (36 +/- 7 mM); and no affinity for l-fucose. Although these functional characteristics essentially confirmed those found in rat intestinal apical membranes, a unique discrepancy was seen between the two systems studied in that the affinity constant for glucose was approximately 40-fold lower in vesicles (K(i) = 0.94 +/- 0.35 mM) than in oocytes. Finally, the transport system responsible for the basolateral efflux transporter of glucose in intestine, GLUT2, did not mediate any significant radiolabeled MI uptake in oocytes, indicating that this transport system does not participate in the basolateral exit of MI from small intestine.     

Effect of size of Trichinella spiralis (Nematode) infections on glucose and ion transport in the rat intestine

An in vivo perfusion technique, using 3 intestinal loops representing the anterior, mid and posterior regions of the rat small intestine, was used to determine intestinal glucose uptake 5 days after infection with Trichinella spiralis. At high levels of infection (3,000 and 6,000 larvae/rat) net glucose absorption by the intestinal mucosa was significantly impaired in all regions of the small intestine when compared to uninfected controls. At low levels of infection (50 larvae/rat) glucose uptake by the mucosa was significantly enhanced in all 3 regions of the small intestine. Intermediate levels of infections (200-1,000 larvae/rat) also enhanced glucose uptake, but only in the anterior regions of the small intestine. When washings from the small intestine of rats infected with 50 larvae/rat were added to the perfusion fluid used on uninfected rats, glucose uptake was also significantly enhanced. These results suggest that at low levels of infection the intestinal lumen contains a metabolite which may affect the mucosal transport of glucose and the related fluxes of H2O, Na+, Cl-, and K+, in the rat intestine. Luminal [H+] and pCO2 decreased from the proximal to distal regions of the small intestine following perfusion; pO2 was significantly decreased in the proximal and distal regions.     

Role of -SH groups in rat sugar intestinal transport in vivo.

The effect of p-chloromercuribenzoic acid (pCMB), either alone or in the presence of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), on the 1 mM galactose absorption by in vivo perfused rat intestine has been studied. At 0.25 mM concentration, pCMB inhibits galactose absorption in about 32% but it does not modify the absorption of this sugar when the transport is blocked by 0.5 mM phlorizin, or that of the non-transportable monosaccharide derivative 2-deoxy-D-glucose. This shows that only the active transport component of galactose absorption is inhibited. A 2 min preexposure period is required for the inhibition to appear. The inhibition was not reversed by washing with saline solution even when it contained 0.5 mM dithioerythritol, 10 mM cysteine or 5 and 10 mM EDTA. The simultaneous exposure to 0.25 pCMB and 0.25 mM DTNB inhibits the total galactose entry in about 50%, an effect higher than the one exerted by each reagent separately and close to the one obtained with 0.5 mM phlorizin. Our results, in vivo, confirm the importance of the thiol groups in the cotransport of Na+ and sugar. As DTNB is an SH-reagent of lesser liposolubility than pCMB, the existence of two populations of sulfhydryl groups related to sugar transport which differ in their location within the brushborder membrane and in accessibility from the intestinal lumen, is suggested.     

Oxyphenisatin derivatives and intestinal glucose and tyrosine absorption

The effect of oxyphenisatin and three other isatin derivatives on glucose and tyrosine absorption is studied in rat small intestine, in vitro. Compounds with free phenolic groups elicit, even at low concentrations, strong inhibition on active transport, while those with the sulfate-esterified phenolic groups show no effect whatsoever. One minute preincubation with 10(-7) M oxyphenisatin is enough to inhibit sugar absorption completely.     

Study of glucose concentrated solution consumption in rats and simulation of glucose distribution along the intestine

Free ingestion of glucose solution (200 or 400 g/l) by Wistar rats, previously starved for 18-20 Hrs, was investigated in two groups of the animals: with intact small intestine (group 1, n = 9), and a shortened small intestine following the Thiry-Wella isolation of its one third proximal part (group 2, n = 9). In the rats of the group 2, the isolated intestinal loops were perfused in chronic experiments with soulutions of different glucose concentrations to estimate a permeability of the pre-epithelial ("unstirred") layer and "true" kinetic constants of glucose active transport. The rate of glusouse ingestion was found to be 1.3-fold as high in the of rats fgroup 1 than in the rats of group 2 (p < 0.01). According to results of mathematical modeling, the rate of glucose ingestion by rats corresponds to glucose concentration in the initial solutions and to the absorbing capacity of the small intestine due to the substrate regulation of gastric emptying. The model predicts that, during free ingestion by rats of 400 g/l (2200 mM) glucose solution, the substrate concentration in the intestinal lumen under steady state conditions hardly exceeds 75 mM. This fact contradicts a recently proposed hypothesis about a facilitated transport mediated by GLUT2 as the main mechanism of glucose absorption in the small intestine under normal conditions.     

Absorption kinetics for leucine, cycloleucine and alpha-aminoisobutyric acid by rat small intestine in vivo

The intestinal absorption kinetics of three neutral amino acids, leucine, cycloleucine and alpha-aminoisobutyric acid, has been studied in rat jejunum in vivo, with luminal perfusion during successive periods, by measuring the passive component and the active transport. The mass-transfer coefficients of the passive process, are similar for the three amino acids and increase with the perfusion rate. The transport component, obtained from the difference between total absorption and passive diffusion, shows saturation kinetics and also increases with the perfusion rate. The apparent Michaelis constants, Km, and the maximal transport rates for the three amino acids have been determined. The Km values are greater than those reported for in vitro studies, a result imputable to greater thickness of the unstirred layers in vivo and to the unequal signification of the constant in both conditions. Passive flux has proved to be an important component for in vivo absorption, even at low substrate concentrations (1-5 mM), so that its evaluation cannot be neglected for the calculation ot the kinetic constants of the mediated transport.     

Effect of phloretin and phloridzin on properties of digestion and absorption in the rat small intestine

Experiments in vitro on everted sacs of rat small intestine have shown that phloretin (an inhibitor of basolatheral glucose GLUT2 transporter) added from mucosal side of the sacs decreases release of glucose from enterocytes into serosal fluid without changing glucose accumulation in tissue of the preparations. Addition of phloridzin (an inhibitor of Na⁺ and glucose co-transporter SGLT1) from mucosal side inhibited both glucose accumulation in the tissue and its release into serosal fluid. Unspecific effects of phloretin and phloridzin on activities of several digestive enzymes (in particular, alkaline phosphatase, amino peptidase, and glycyl-L-leucine dipeptidase) has been revealed in homogenates of the rat small intestine mucosa. In chronic experiments on rats, absorption of glycine from the isolated small intestinal loop was inhibited in the presence of phloretin in perfusate. The obtained results indicate that the experimental approach of inhibition of glucose absorption by phloretin used from mucosal side in vitro appears to give a significant overestimation of contribution of facilitated diffusion (with participation of the GLUT2 transporter inserted in the apical enterocyte membrane) to glucose transport across this membrane. Thus, the role of the GLUT2 transporter in the mechanism of glucose absorption in the small intestine under its physiological conditions does not seem to be as great as it is thought by the authors of the recently proposed hypothesis.     

A revision of current data and views on membrane hydrolysis and transport in the mammalian small intestine based on a comparison of techniques of chronic and acute experiments: experimental re-investigation and critical review

Literature data and the results of our investigations using both generally accepted and original perfusion techniques of the isolated loop of the rat small intestine in in vivo experiments are reviewed. Significant differences in the functioning of the small intestine under conditions of acute and chronic experiments are revealed. It has been established that in chronic experiments as compared to acute ones: (a) the absorption of glucose, galactose, fructose and glycine is 2-5 times higher; (b) Kt as well as Jmax values of the transport of these nutrients differ considerably; (c) Na+-independent mechanism of glucose and glycine transport predominates; (d) higher rates of membrane hydrolysis and more effective interactions between enzyme and transport systems of the enterocyte brush border membranes are observed; (e) functional characteristics of the small intestine affected by various experimental factors are more stable. The conclusion is made that it is necessary to revise current views of the scale and regularities of digestive-transport processes in the small intestine under physiological conditions. The importance of the suggested approaches for general and comparative physiology and biochemistry is discussed.     

Isoosmotic transport of fluid across the hamster small intestine in the presence of phlorizin-induced inhibition of sugar transport.

Experiments were performed to investigate whether the fluid transported across the small intestine is isoosmotic with the mucosal solution when the active transport of glucose is partially inhibited. Everted hamster mid small intestine was incubated in one of the following four mucosal solutions: (1) Isotonic control, Krebs-Ringer bicarbonate solution containing 10 mM glucose (KRBSG), (2) Isotonic with phlorizin, KRBSG + 5X10-5 M phlorizin, (3) Hypertonic control, KRBSG + 50 mM mannitol, (4) Hypertonic with phlorizin, KRBSG + 50 MM mannitol + 5x10-5 M phlorizin. The serosal surface of the intestine was not bathed. Results indicate that the transported fluid was always isoosmotic with any of the mucosal solutions used. When the mucosal solution was made hypertonic with mannitol, the concentration of glucose and electrolytes in the absorbate increased, and as a result, the absorbate became hypertonic and isoosmotic with the mucosal solution. The presence of phlorizin either in the isotonic or in the hypertonic mucosal solution decreased the glucose concentration of the absorbate, but the transported fluid became isoosmotic with the mucosal solution due to a higher concentration of Na, K, and their associated anions. Phlorizin caused a decrease in the transmural potential difference. In spite of this, the presence of this glucoside in the mucosal solution increased the transport of sodium in relation to glucose transport. It is suggested that, at the concentrations used, phlorizin inhibits sodium movement through the electrogenic pathway, but increases the transport of this ion through the non-electrogenic route. This increase in neutral sodium transport seems to compensate for the low concentration of glucose in the absorbate, so that the absorbate becomes isoosmotic with the mucosal solution whether the latter is isotonic or hypertonic. It is suggested further that isoosmotic transport of fluid is an inherent property of the small intestine and that there may be an osmoregulatory mechanism in the gut which controls this process.     

Effect of luminal Na+ on the kinetics of intestinal absorption of sugars in vivo

The effect of sodium concentration on the absorption kinetics of glucose, galactose and 3-o-methyl-glucose in rat and hamster jejunum in vivo has been studied. In consecutive 1 min periods the total absorption and absorption in presence of 0.5 mM phlorizin were measured. The difference between them was taken as the active transport rate. The perfusion rate value was 5.6 ml X min-1 and sugar concentrations in the perfusion solution ranged from 1 to 10 mM. The results for the different sodium concentrations show a nearly common Vmax for the same sugar and animal species, while the apparent KT values increase when the sodium concentration in the lumen decreases, mimicking a pure affinity-type activation system. The absorption of sugar when solutions without Na+ are perfused, is greater than that entering passively in the presence of phlorizin. An explanation may be that appreciable amounts of endogenous Na+ find their way to the intestinal lumen in favour of the gradient, making Na+-sugar cotransport possible.     

Geometric peculiarities of intestinal surface and efficiency of coupling between membrane hydrolysis and transport of nutrients

Kinetics of the membrane hydrolysis of maltose and of the absorption of the released glucose in the isolated loop of rat small intestine has been examined in a wide range of maltose concentrations (25–200 mM) under the conditions of chronic experiments. The processes studied were simulated by means of mathematical models using two approximations of the villous surface of intestinal epithelium: (i) smooth flat surface with adjoining pre-epithelial (“unstirred”) layer and (ii) folded surface with “unstirred” layer between the folds and partly above them. The results of modeling matched well the experimental data in the whole range of maltose concentrations only in the case of the folded surface. A model with this approximation predicts a closer coupling between maltose hydrolysis and absorption of released glucose as well as a lower glucose concentration in the intestinal lumen than in the case of a flat surface. We conclude that in order to evaluate correctly a relative role of various mechanisms of glucose transport across intestinal epithelium under normal conditions, one should take into account the pre-epithelial layer of the small intestine and geometric peculiarities of its epithelial surface.