[FREE CONSUMPTION OF GLUCOSE SOLUTION BY RATS AS A CRITERION FOR EVALUATION ITS ABSORPTION IN THE SMALL INTESTINE (Experimental study and mathematical modeling)].

The aim of the work is to analyze the relationship between consumption of glucose solution by rats and its absorption, and to use this fact for assessment of the absorptive capacity of the small intestine in non anesthetized animals in vivo. Consumption of glucose solution (200 g/l) by fasted rats was recorded in the control, and after administration of phloridzin--inhibitor of glucose active transport- or 3 hours after the restriction stress. On the mathematical model we studied the relative role of factors that can influence the temporal dynamics of glucose consumption by rats. The rate of glucose consumption was observed being decreased in the presence of phloridzin (1 mM), and be increased after the stress. The results of modeling are consistent with the experimental data and show that the rate of consumption of glucose solutions considerably more depends on the transport activity of the small intestine than on glucose concentration in the solution, or on the substrate regulation of the stomach emptying. Analysis of dynamics of consumption of glucose solution by intact rats may be considered as one of promising approaches to assessing the absorptive capacity of the small intestine under natural conditions.

[The interaction between SGLT1 or GLUT2 glucose transporter and the cytoskeleton in the enterocyte as well as Caco2 cell during hexose absorption].

The distribution of cytoskeleton elements (microtubules and actin filaments) and SGLT1 or GLUT2 glucose transporter in enterocyte of rat intestine and Caco2 cell during hexose absorption has been considered. The alteration of SGLT1 and GLUT2 transporter distribution in absorptive cell of intestine villus depending on maltose concentration has been determined using the confocal microscope. The colocalization of the transporters and actin has been revealed. The increase of vesicles number close to microtubules in the apical part of cell during absorption of high hexoze concentration has been found by electron microscope. The fact together with the transporter and actin as well as actin and α-tubulin colocalizations can prove the participation of cytoskeleton elements in glucose transporter movement to apical membrane of the cells studied.

[Glucose absorption in the rat small intestine in vivo after various levels of local substrate load].

In order to evaluate relative roles of various mechanisms of glucose transport in the small intestine at high substrate loads in chronic experiments on rats, we investigated kinetics of glucose absorption in isolated part (-20 cm) of the intestine after its perfusion for 6 and 14 days during 1.5 h per day with 125 mM glucose solution (gr. 1--increased substrate load) or during 45-60 min per day with 25 mM glucose solution (gr. 2--reduced substrate load). The results of the experiments were analyzed by means of mathematical simulation. It was found that in the rats of gr. 1 the regular substrate load was more effective in maintaining a high level of glucose absorption in the isolated part of the intestine. Adaptation of glucose absorption to the increased local glucose load occurs due to enhancement of the secondary active transport via SGLT1. This component in many times exceeds the "unsaturated" component of glucose absorption, which is mainly determined by the facilitated diffusion via GLUT2, both at high and low glucose concentrations in the intestinal lumen.

[Responses of peptide hydrolases of the small and large intestines in rats on the administration of antibiotics].

Effects of antibiotics on the structure and functional state of the intestine are not clear. We investigated some structural parameters of the small and large intestine, and activities of two intestinal peptide hydrolases in rats after administration of ampicillin and metronidazole during 3 and 5 days. After 3 days of antibiotic administration a decrease in the weight of mucosa in the small intestine, accompanied with a reduction in the villous height and width in this part of the intestine, and in the weight ofmucosa in the colon occured. At the same time the number of goblet cells in the small intestinal epithelium was increased. Specific activities of aminopeptidase M, and glycyl-L-leucine dipeptidase (micromol/min per g) in the mucosa of the small intestine were increased, and the total activities (micromol/min calculated per a part of the intestine) of the same enzymes did not change. The administration of antibiotics for 5 days resulted in increase of specific activity ofaminopeptidase M in the mucosa of the proximal part of the small intestine. In the chyme of the small intestine and colon, activities of the same enzymes (micromol/min calculated per a part of the intestine) were increased on the third and fifth days of the antibiotic administration. Thus, the application ofampicillin and metronidazole within 3-5 days causes a disturbance of the structural and functional parameters in the small and large intestines, which is most pronounced on the third day of the drug administration.

[Caco 2 cell culture as intestinal epithelium model for hexose transport studying].

Distribution of SGLT1 and GLUT2 hexose transporters as well as that of fibrillar actin and tight junction proteins in cultured Caco2 cells incubated in medium with different hexose concentrations has been considered. Glucose absorption by the cells from incubation medium has been determined. Fibrillar actin was concentrated in the microvilli and closely to tight junction. The actin distribution was not dependent on the glucose concentration. There was no SGLT1 association with brush border actin and the transporter localization was not dependent on the concentration of hexose. GLUT2 was localized in the basal part of Caco2 cells after low concentration hexose load (2.5 mM). The transporter was colocalized with microvilli actin in the apical part of the cells after high concentration hexose load (25 mM). The tight junction proteins, occludin and claudin 1, 3, 4 were not dependent on glucose concentration. Claudin 2 was not detected in Caco2 cells. Caco2 cell culture can be used as a model for studying of hexose transport in small intestine epithelium.

[Glucose absorption from mono- and oligosaccharide solutions in the rat small intestine in vivo].

The rates of maltose and maltotriose hydrolysis, and glucose absorption in the isolated loop of the rat small intestine, perfused by isocaloric solutions of the above substrates, were examined in chronic experiments. In all the experiments, the rates of glucose absorption from the solutions of maltose and maltotriose (M- and MT-glucose, respectively) were almost the same as those from the isocaloric solutions of free glucose (G-glucose). The rate of water absorption in the isolated intestinal loop was significantly higher under the perfusion with maltose (100 mM) and maltotriose (66.6 mM) solutions than under the perfusion with equivalent glucose solutions. The results of mathematical simulation, in which absorptive surface of the small intestine was approximated as a folded surface with an adjoining zone of diffusion, were in a good agreement with the experimental data. The model showed that in the range of physiological concentrations of the substrates their transfer across the pre-epithelial layer by water flux seemed to play a minor role as compared with a transfer by diffusion. According the results obtained, the most significant factors that influence the efficiency of coupling between hydrolysis and absorption of nutrients, are following: a complex geometry of intestinal surface, the pre-epithelial diffusion layer, the rate of water absorption (secretion) in the intestine.

[Comparative analysis of SGLT1 and GLUT2 transporter distribution in rat small intestine enterocytes and Caco2 cells during hexose absorption].

SGLT1 and GLUT2 hexose transporter distribution into enterocytes of small intestine isolated loop and Caco2 cell culture after absorption of high and low hexose concentrations has been considered. SGLT1 was found along intestine villus edge in isolated loop. After high concentration hexose load GLUT2 appeared to be situated in the apical parts of enterocytes. It is evident that GLUT2 participates in hexose transport across apical membrane. Cultured Caco2 cells form microvilli and cell junction complex typical for enterocyte. Glucose and galactose absorption by the cells from incubation medium has been observed. SGLT1 transporter is situated in the apical parts and around the nuclens of Caco2 cells and combined into globules. After low concentration hexose load, CLUT2 transporter is localized in the basal parts of Caco2 cells. Caco2 cell culture can be used as a model for studying of hexose transport in small intestine epithelium.

[Evaluation of the role of the peptide transport system in absorption of dipeptides in the rat small intestine in chronic experiments in vivo].

Hydrolysis and absorption of glycylgycine and glycyl-L-leucine as well as absorption of glycine and leucine were studied in chronic experiments on rats with their isolated small intestine loop. Values of the true kinetic constants (with taking into account effect of the preepithelial layer) were determined to be as follows: 1) Kt = 46.7 +/- 4.0 and 2.15 +/- 0.59 mM; Jmax = 0.74 +/- 0.15 and 0.16 +/- 0.03 micromol x min(-1) x cm(-1) (for the transport of free glycine and leucine, respectively); 2) Kt = 4.4 +/- 0.6 and 4.8 +/- 0.9 mM, Jmax = 0.24 +/- 0.02 and 0.23 +/- 0.02 micromol x min(-1) x cm(-1) (for the transport of glycylgycine and glycyl-L-leucine, respectively); 3) Km = 5.4 +/- 1.0 and 38.2 +/- 4.4 mM, Vmax = 0.09 +/- 0.02 and 0.24 +/- 0.07 micromol x min(-1) x cm(-1) (for membrane hydrolysis of these dipeptides, respectively). According to our calculations, in the wide range of the initial glycylgycine concentrations (2.5-40 mM) the part of the peptide component in its total absorption accounts for 0.77-0.80. In the case of glycyl-L-leucine the part of the peptide component in the total glycine absorption decreases from 0.89 to 0.84, while in the total leucine absorption--from 0.86 to 0.71, the initial dipeptide concentration rising from 5 to 40 mM. The obtained results show that the peptide component prevails in absorption of the studied dipeptides in the rat small intestine, but its role is much lesser that what many authors believe. In the case of glycyl-L-leucine, the peptide component can achieve saturation in the range of high substrate concentrations, its part decreasing essentially to become compared with absorption of free amino acids formed as a result of the dipeptide membrane hydrolysis.

[Structural-functional analysis of diffusion in glucose absorption by rat small intestine enterocytes].

To elucidate mechanisms providing transport of sugars across intestinal epithelium, on taking into account the current hypotheses (active transport, participation of paracellular transport and passive component of transcellular transport), it was important to reveal structural changes of tight junctions and distribution of the carriers of facilitated diffusion of GLUT2 and protein kinase C during absorption of glucose. On using confocal and electron microscopy, ultrastructural and immunocytochemical studies of enterocytes after perfusion of isolated rat small intestine fragment with 75 mM glucose (chronic experiment) have shown: 1) fluorescent labels of transporter GLUT2 and PKCbetaII are located in the apical area of enterocytes situated at the upper half of the villus. Antibodies against GLUT2, conjugated with gold, are revealed at the microvilli or apical membrane and in the area of terminal network; 2) no ultrastructural changes of the tight junction are detected on ultrathin sections and freeze--fracture replics. At the same time, fluorescent and gold labels against actin are concentrated in the vicinity of the lateral membrane in the tight junction area. The results obtained can serve a confirmation of a hypothesis that at high glucose concentrations GLUT2 participates in its transfer across the apical membrane.

[The role of facilitated diffusion in glucose transport across the apical membrane of enterocytes].

In chronic experiments on Wistar rats, glucose and galactose absorption in the isolated loop of the small intestine considerably decreased in presence of both phloridzine am phloritine (inhibitors of the glucose transporters SGLT1 and GLUT2). The load of the isolated loop with glucose or galactose solutions scarcely influenced the absorption of 2-deoxi-D-glucose (substrate for GLUT2). According to the immunocytochemical analysis by means of confocal microscopy, after the load of the isolated loop with glucose (75 mM) the labels to GLUT2 and proteinkinase C (PKC betalI) were concentrated mainly in the apical part of the enterocytes, whereas after the load with the Ringer solution--in the basal part of the enterocytes. It was shown on the mathematical model that the part of the facilitated diffusion in the total glucose absorption was considerably lesser in comparison with the active transport mediated by SGLT1. Thus the findings support the hypothesis about a recruitment of the transporter GLUT2 into the apical membrane of the enterocytes and its involvement in glucose transfer across this membrane. However, under natural conditions, the active transport is the main mechanism of glucose absorption, whereas the facilitated diffusion plays a certain role only at high carbohydrate loads.

[Study of glucose concentrated solution consumption in rats and simulation of glucose distribution along the intestine]. / Issledovanie potrebleniia krysami kontsentrirovannykh rastvorov gliukozy i modelirovanie ee raspredeleniia vdol' kishki.

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.

[Structural and functional analysis of glucose adsorption at high maltose concentrations in the rat small intestine in vivo]. / Strukturno-funktsional'nyi analys mekhanizmov vsasyvaniia gliukozy pri vysokikh kontsentratsiiakh mal'tozy v tonkoi kishke krys 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.

[Kinetic analysis of glycine and glycylglycine absorption in rat small intestine in chronic experiment]. / Kineticheskii analiz vsasyvaniia glitsina i glitsilglitsina v tonkoi kishke krys v usloviiakh khronicheskogo opyta.

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.

[Kinetic parameters of maltose hydrolysis and glucose intake in the rat small intestine in a chronic experiment]. / Kineticheskie parametry gidroliza mal'tozy i vsasyvaniia gliukozy v tonkoi kishke krys v khronicheskikh opytakh.

"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.

[Mechanisms of glucose absorption at a high carbohydrate level in the rat small intestine in vivo]. / Mekhanizmy vsasyvaniia gliukozy v tonkoi kishke krys in vivo pri vysokoi kontsentratsii uglevodov.

At low maltose concentations, the rates of maltose hydrolysis and glucose absorption increased with increasing substrate concentration in the infusate. A significant fluid secretion occurred in the isolated intestinal loop perfused with hypertonic maltose solution, but water fluxes were close to zero in case of almost isotonic perfusion solutions. The findings suggest that the active transport remains a main mechanism of glucose absorption at super-high maltose concentration as well. The rates of maltose hydrolysis and released glucose absorption in the isolated intestinal loops perfused with super-high maltose concentration, enhance mainly due to an increase of effective digestive-absorptive surface of the villi and, in part, to substrate diffusion across the intestinal epithelium rather than paracellular solvent drag.

Hydrolysis-dependent absorption of disaccharides in the rat small intestine (chronic experiments and mathematical modeling).

In order to throw light on the mechanisms responsible for the enzyme-dependent absorption of disaccharides membrane hydrolysis of maltose and trehalose and the absorption of glucose (free and that derived from disaccharides) were studied in isolated loops (20 cm) of the rat small intestine in chronic experiments. The rates of glucose absorption were 0.26-0.81 micromol x min(-1) x cm(-1) when the loop was perfused with a 12.5 to 75.0 mmol/l free glucose solution, which is only insignificantly higher than the rates observed during perfusion with equivalent maltose solutions. The coupling coefficient (the ratio of glucose absorption rate to the rate of disaccharide hydrolysis) decreased from 0.90 to 0.60 with the increasing maltose concentrations in the infusate from 6.25 to 37.5 mmol/l, but remained unchanged (approximately 0.95) within the same range of trehalose concentrations. The permeability of the pre-epithelial barrier was equivalent to that of unstirred water layer of less than 40 microm thickness. Fluid absorption was within the range of 0.73-2.55 microl x min(-1) x cm(-1), and it showed a correlation with the rates of glucose absorption. The results agree with a model developed on the assumption that free glucose and that released from disaccharides share the same membrane transporters. It could be concluded that a close coupling of disaccharide hydrolysis with derived glucose absorption in chronic experiments is achieved mainly due to a high activity of glucose transporters, which are presumably not associated with membrane disaccharidases. The transcellular active transport is a predominant mechanism of disaccharide-derived glucose absorption under conditions close to physiological.

[Effect of protein deficiency in the female rat diet during pregnancy and lactation on the activity of the membrane and soluble forms of digestive enzymes in the offspring small intestine]. / Vliianie nedostatochnosti belka v pitanii krys-samok v techenie beremennosti i laktatsii na aktivnost' membrannoi i rastvorimoi form pishchevaritel'nykh fermentov tonkoi kishki potomstva.

Protein deficiency in female rats diet during pregnancy and lactation resulted in deceleration of induction of sucrase both forms in the jejunum and ileum; in acceleration of induction of the maltase membrane from in the jejunum; and in suppression of the lactase membrane form in the ileum; in earlier forming of the adult-type distribution of activity of the membrane form of intestinal alkaline phosphatase and in a decrease in activity of the enzyme soluble form. The findings are corroborated by a suppression of activities of the membrane and soluble forms of the small intestine digestive enzymes in 30-day old rat pups fed with a control (adequate) ration starting 21 days after the birth.

[Analysis of rat enterocyte ultrastructure during glucose absorption]. / Analiz ul'trastruktury énterotsitov krysy pri vsasyvanii gliukozy.

Electronmicroscopic study of rat enterocytes under glucose load (10-40 mM) has shown some changes of their structure: aggregations of intramembrane particles of the apical membrane in the microvilli region, the dilitation of lateral intercellular spaces below tight junction, the condensation of actin near tight and intermediate junctions. The presence of these changes and almost absolute absence of destructions in tight junctions organization indicate that the main pathway of the isotonic fluid containing glucose across leaky epithelium of rat small intestine is a transcellular one.

[Glucose and galactose absorption in the small intestine of rats in vivo]. / Vsasyvanie gliukozy i galaktozy v tonkoi kishke krys in vivo.

In mathematical models, competition between glucose and galactose in the course of their active transport across the apical membrane of the erythrocytes, was studied. Both substances seem to share the same means of transmembrane transport, although the affinity of glucose in 6-fold higher.