Christiane Malo

Influence of Epidermal Growth Factor on the Development of Suckling Mouse Intestinal Mucosa

This investigation was undertaken to study the influence of epidermal growth factor on the development of intestinal brush border hydrolytic activities and on the deoxyribonucleic acid synthesis in suckling mice. A premature appearance of sucrase activity was elicited in mice after a single injection of epidermal growth factor at 8 days of age or after one injection per day during 3 days, the level of sucrase activity being dependent of the amount of epidermal growth factor injected (1 or 4 μg body wt · day). Trehalase, glucoamylase, lactase, alkaline phosphatase, and γ-glutamyltranspeptidase activities were increased along the entire small intestine after epidermal growth factor treatment. On the other hand, endoplasmic reticulum membrane-bound glucose-6-phosphatase activity was unaffected by the polypeptide hormone. The effects of a single injection or repeated injections of 4 μg epidermal growth factor/g body wt · day on the deoxyribonucleic acid synthesis in the proximal and distal intestine (sectioned into thirds) as well as in the proximal colon have been determined. While a single injection of epidermal growth factor did not affect the proliferation of the intestinal epithelium, one injection per day during 3 days provoked a significant increase of 3H-thymidine incorporation into deoxyribonucleic acid and a significant enhancement of the mitotic activity. It was concluded that epidermal growth factor is able to influence the maturation and the proliferation of the enterocytes in suckling mouse and that the two phenomena appear to be differentially sensitive to epidermal growth factor in the present conditions.

Analysis of kinetic data in transport studies: New insights from kinetic studies of Na+-d-glucose cotransport in human intestinal brush-border membrane vesicles using a fast sampling, rapid filtration apparatus

SummaryUsing the fast sampling, rapid filtration apparatus (FSRFA) recently developed in our laboratory (Berteloot et al., 1991.J. Membrane Biol.122:111–125), we have studied the kinetic characteristics of Na+-d-glucose cotransport in brush-border membrane vesicles isolated from normal adult human jejunum. True initial rates of transport have been determined at both 20 and 35°C using a dynamic approach which involves linearregression analysis over nine time points equally spaced over 4.5 or 2.7 sec, respectively. When the tracer rate of transport was studied as a function of unlabeled substrate concentrations added to the incubation medium, a displacement curve was generated which can be analyzed by nonlinear regression using equations which take into account the competitive inhibition of tracer flux by unlabeled substrate. This approach was made imperative since at 20°C, in the presence of high substrate concentrations or 1mm phlorizin, no measurable diffusion was found and the resultant zero slope values cannot be expressed into a classicalv versus S plot. All together, our results support the existence of a single Na+-d-glucose cotransport system in these membranes for which Na+ is mandatory for uptake. This conclusion is at variance with that of a recent report using the same preparation (Harig et al., 1989.Am J. Physiol.256:8618–8623). Since the discrepancy seems difficult to resolve on the consideration of experimental conditions alone, we have determined the kinetic parameters ofd-glucose transport using one time point measurements and linear transformations of the Michaelis-Menten equation, in order to investigate the potential problems of such a widely used procedure. Comparing these approaches, we conclude that: (i) the dynamic uptake measurements give a better understanding of the different uptake components involved; (ii) it does not matter whether a dynamic or a one time point approach is chosen to generate the uptake data provided that a nonlinear-regression analysis with proper weighting of the data points is performed; (iii) analytical procedures which rely on linearization of Michaelian process(es) are endowed with a number of difficulties which make them unsuitable to resolve multicomponent systems in transport studies. A more general procedure which uses a nonlinear-regression analysis and a displacement curve is proposed since we demonstrate that it is far superior in terms of rapidity, data interpretation, and visual information.

Activation of amino acid diffusion by a volume increase in cultured kidney (MDCK) cells

SummaryWhen MDCK cells are cultured in MEM, they maintain a high concentration of three amino acids: glutamate (25mm), taurine (19 mm) and glycine (9 mm). With incubation of the cells in hypotonic media, the contents of these amino acids measured by HPLC are reduced in different time courses: taurine decreases most rapidly, followed by glutamate and glycine. All these losses are Na+ independent. To determine the transport mechanism activated by the hypotonic media, increasing external concentrations reaching 60 mm for nine different amino acids in Na+-free media were tested separately. For the five neutral (zwitterionic) amino acids, taurine, glycine, alanine, phenylalanine and tryptophan, cell contents increased linearly with external concentrations in hypotonic media, whereas in isotonic media only a slight rise was observed. The two anionic amino acids, glutamate and aspartate, were also increased linearly with their external concentrations in hypotonic media, but the changes were lower than those found for neutral amino acids. The presence of a negative membrane potential was responsible for this behavior since, using a K+ hypotonic medium which clamps the potential to zero, the glutamate content was found to increase linearly with an amplitude similar to the one observed for neutral amino acid. When external concentrations of two cationic amino acids, arginine and lysine, were increased in hypotonic media, only a small change, similar to that in isotonic media, was observed. These results indicate that a diffusion process for neutral and anionic amino acids is activated by a volume increase and it is suggested that an anion channel is involved.

Glucagon-like peptide 2 stimulates intestinal nutrient absorption in parenterally fed newborn pigs.

Objectives: Parenteral nutrition is a critically important intervention for children with intestinal dysfunctions. However, total parenteral nutrition (TPN) with no enteral feeding is associated with small intestine atrophy and malabsorption, which complicate the transition to enteral nutrition. The objective of the present study was to evaluate the therapeutic potential of the intestinotrophic peptide glucagon-like peptide 2 (GLP-2), which reduces TPN-associated atrophy and maintains nutrient absorption in adult rats, for preventing nutrient malabsorption in neonates receiving TPN. Methods: Term pigs obtained by cesarean delivery received from birth TPN alone (TPN; n = 7) or TPN with GLP-2 (25 nmol · kg−1 · d−1; GLP-2; n = 8) or were fed sow milk enterally (n = 7). The small intestine was removed on postnatal day 6 to measure morphological responses and absorption of glucose, leucine, lysine and proline by intact tissues and brush border membrane vesicles and to quantify the abundances of mRNA and protein for enterocyte glucose transporters (SGLT-1 and GLUT2). Results: Relative to TPN alone, administration of GLP-2 resulted in small intestines that were larger (P < 0.01), had greater abundances of mRNA and protein for SGLT-1, but not for GLUT2, and had higher capacities to absorb nutrients (P < 0.01). Moreover, the intestines of GLP-2 pigs were comparable in size and absorptive capacities with those of pigs fed sow milk enterally. Conclusions: Providing GLP-2 to neonates receiving TPN prevents small intestine atrophy, results in small intestine absorptive capacities that are comparable to when nutrients are provided enterally and may accelerate the transition from TPN to enteral nutrition.

Intestinal brush-border membrane enzyme activities and transport functions during prenatal development of pigs

Enzyme activities and rates of leucine and glucose uptake were measured using brush-border membrane vesicles prepared from the small intestine of 7-, 8-, 10-, and 12-week fetal (43, 49, 61, and 74% of gestation) and unsuckled, neonatal pigs. Lactase was detected in 7-week fetuses, with a large increase in activity between 10 weeks of gestation and birth. gamma-Glutamyltranspeptidase activity was stable throughout gestation, whereas sucrase activity was not detected. Active L-leucine uptake was already present at 7 weeks of gestation, with an increasing distal-to-proximal gradient observed at birth. D-glucose uptake was low at 7 weeks, but by 8 weeks it exhibited a typical overshoot phenomenon and established a decreasing proximal-to-distal gradient by 12 weeks. D-glucose uptake at all ages was directly related to incubation temperature, but less so for 7- and 10-week fetuses. By 12 weeks strict Na(+)-dependency of D-glucose uptake was observed along the entire length of the small intestine. Kinetic analysis of Na(+)-D-glucose cotransport showed a shift from the presence of both high- and low-affinity systems at 8 weeks of gestation to a single high-affinity Michaelian component at birth. In light of similarities with human fetuses, the pig may be a valuable model for studying development of intestinal transport during gestation, particularly during the final trimester, when availability of human tissue is limited.

The biosynthesis of intestinal sucrase-isomaltase in human embryo is most likely controlled at the level of transcription

Although sucrase-isomaltase appears in the small intestine at quite different stages of development in man as compared with most mammals, we find that in human embryo also the appearance of sucrase-isomaltase mRNA closely parallels that of sucrase and isomaltase activities, as we have previously found to be the case in baby rabbits. Also, in the proximal-distal gradient of human embryonic intestine (proximal small intestine greater than distal small intestine greater than colon) the levels of these enzyme activities and those of the corresponding mRNA correlate closely. Finally, glucocorticosteroid treatment of a human colon carcinoma cell line (Caco-2) in vitro or of baby rabbits in vivo leads to a parallel increase of both sucrase and isomaltase activities and of sucrase-isomaltase mRNA. We conclude that in man also, in spite of the different timing in development, the biosynthesis of sucrase-isomaltase is most likely to be controlled at the level of transcription or perhaps of the mRNA stability.

Multiple Pathways for Amino Acid Transport in Brush Border Membrane Vesicles Isolated From the Human Fetal Small Intestine

The present study was undertaken to identify the different amino acid transport pathways present in the human small intestine during the early gestational period. The uptake time courses of neutral (L-leucine, L-alanine, L-methionine), acidic (L-glutamic and D-aspartic acids), basic (L-lysine), and imino (L-proline) acids have been studied in brush border membrane vesicles isolated from both proximal and distal parts of the human fetal small intestine. Both Na(+)-dependent and Na(+)-independent uptake pathways have been identified all along the small intestine. The Na(+)-dependent systems are as follows: (a) the NBB system for neutral amino acids such as L-leucine and L-alanine; (b) the PHE system for L-methionine; (c) the x-ag system for L-glutamic and D-aspartic acids; and (d) the IMINO system for L-proline. The Na(+)-independent pathways are represented by the L system for most of the neutral amino acids and maybe L-proline and by the basic amino system y+ for L-lysine uptake. These results demonstrate that the different uptake pathways for transport of amino acids are present in the human fetal intestine and that their characteristics in terms of Na+ requirement and proximodistal activity gradient are already established in the early stages of the human development.

Separation of two distinct Na+/d-glucose cotransport systems in the human fetal jejunum by means of their differential specificity for 3-O-methylglucose

Based on kinetic arguments, we have recently proposed the existence of two distinct Na+/D-glucose cotransporters in brush-border membrane vesicles isolated from the human fetal jejunum (Biochim. Biophys. Acta 938 (1988) 181-188). In order to further test this hypothesis, inhibition studies of the zero-trans influx of substrate have been performed under Na(+)-gradient and voltage-clamped conditions. Initial rates of D-glucose uptake were totally abolished by D-glucose, D-galactose, alpha-methylglucose and phlorizin while 3-O-methylglucose and phloretin induced only a 65% inhibition even at the highest concentrations used. The residual activity of D-glucose uptake is thus compatible with substrate flux through a low-affinity transport system which is insensitive to phloretin and does not accept 3-O-methylglucose as substrate. This substrate specificity has been used to separate kinetically the two putative pathways for glucose transport. The data obtained are compatible with the existence of the following two systems: (i) a low-affinity, high-capacity system with a Km of 4.7 mM and a Vmax of 22 nmol/min per mg of protein, and; (ii) a high-affinity, low-capacity system with a Km of 0.57 mM and a Vmax of 10.7 nmol/min per mg of protein. These data thus demonstrate clearly the existence of two distinct Na(+)-dependent D-glucose carriers in the human jejunum during the early gestation period since these systems can be differentiated not only by their kinetic properties but also by their differences in both substrate and inhibitor specificities.

Kinetic evidence for heterogeneity in Na+-ld-glucose contransport systems in the normal human fetal small intestine

Zero-trans kinetic studies of Na+-D-glucose cotransport have been performed under voltage-clamped conditions in brush-border membrane vesicles isolated from both jejunum and ileum of 17-20-week-old normal human fetuses. Varying glucose concentrations in the incubation medium led to curvilinear Eadie-Hofstee plots in the jejunum only, thus suggesting the presence of both high-affinity, low-capacity (Km 0.37 mM; Vmax 8.3 nmol/min per mg protein) and low-affinity, high-capacity (Km 4.2 mM; Vmax 30.9 nmol/min per mg protein) systems in the proximal small intestine, and of a single carrier (Km 1.2 mM; Vmax 4.9 nmol/min per mg protein) in the distal small intestine. Sodium activation curves provide further evidence for heterogeneity in glucose transport systems in the fetal small intestine: Hill coefficients of 2 and 1 were found for the jejunal high-affinity and ileal systems, and for the jejunal low-affinity system, respectively. It is concluded that there is early differentiation of a functional heterogeneity in glucose transport capacity along the human fetal small intestine.

Proximo-distal gradient of Na+-dependent D-glucose transport activity in the brush border membrane vesicles from the human fetal small intestine.

Brush‐border membrane vesicles were isolated from the jejunum and ileum of 17–20‐week‐old normal human fetuses and found to be highly enriched in sucrase activity with less than 5% contamination by basolateral membranes. Time course studies of D‐glucose uptake clearly showed a transient, phlorizin‐sensitive, and Na+‐dependent accumulation of sugar into these vesicles. Higher maximum overshoot values and initial rates of D‐glucose uptake were recorded in jejunal as compared to ileal vesicles while low substrate binding to the membranes, identical intravesicular volumes and equivalent dissipation of the Na+‐gradient were found in the two preparations. It was concluded that a fully functional Na+‐D‐glucose cotransport system is present with a proximo‐distal gradient of activity during the early gestation period.