Caco-2 cells, which express spontaneous enterocytic differentiation at confluency, is one of the most relevant in vitro models for the study of differentiation and regulation of intestinal functions. However, these cells are normally cultured in the presence of 15-20% serum which renders extremely complex the identification of the factors involved in the regulation of both proliferation and differentiation. This study has been devoted to the establishment of chemically defined culture conditions which can sustain growth and differentiation of Caco-2 cells. The replacement of serum by ITS (insulin, transferrin, and selenium) allowed for normal structural and functional differentiation of cells as revealed by the establishment of cell polarity and the expression of brush-border membrane enzyme markers (sucrase, maltase, lactase, alkaline phosphatase, gamma-glutamyltransferase, aminopeptidase N, and dipeptidyl-dipeptidase IV), although the levels of sucrase activity were lower in ITS-supplemented medium. Coating petridishes with either type IV collagen or basement membrane proteins (Matrigel) did not improve the differentiation of cells, brush-border membrane enzyme activities being, in fact, lower when the cells were grown on these substrata. When triiodothyronine (T3, 5 x 10(-8) M) was added to the ITS-supplemented medium, disaccharidase and alkaline phosphatase activities were significantly increased while gamma-glutamyltransferase activity was diminished by T3 and stimulated by epidermal growth factor (1.6 x 10(-6) M). On the other hand, hydrocortisone (HC, 10(-6) M) did not modify disaccharidase and peptidase activities. These data clearly show that Caco-2 cells can be maintained in serum-free medium and that this system allows the study of the factors involved in the regulation of the differentiation of enterocyte in vitro.

The uptake of L-ascorbate (vitamin C) and its oxidized form, dehydro-L-ascorbic acid (DHAA), was evaluated in brush border membrane vesicles isolated from adult human duodenum, jejunum and ileum. Ascorbate was taken up along the entire length of the small intestine with a threefold higher initial uptake rate in distal than proximal segments. Ascorbate uptake was Na(+)-dependent, potential-sensitive and saturable (K(m), 200 micromol/L), whereas DHAA transport involved facilitated diffusion (K(m), 800 micromol/L). Pharmacologic experiments were conducted to characterize further these transport mechanisms. DHAA uptake was not mediated by the fructose carrier GLUT5, the uridine transporter or the 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive anion exchanger of the apical membrane. DIDS and sulfinpyrazone, an inhibitor of the urate/lactate exchanger, both significantly reduced the initial rate of ascorbate uptake. Acidic pH inhibited ascorbate uptake, and this effect was not due to a transmembrane proton gradient. Increasing concentrations of glucose in the transport media also significantly inhibited ascorbate uptake, but no effect of glucose was seen when glucose internalization was blocked by phlorizin. Preloading the vesicles with glucose inhibited ascorbate uptake similarly, indicating that glucose interferes with the ascorbate transporter from the internal side of the membrane. The results of this study suggest that DHAA crosses the apical membrane by facilitated diffusion, whereas ascorbate transport is a Na(+)-dependent, electrogenic process modulated by glucose.

Using 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 degrees C using a dynamic approach which involves linear-regression 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 degrees C, in the presence of high substrate concentrations or 1 mM phlorizin, no measurable diffusion was found and the resultant zero slope values cannot be expressed into a classical v 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 of D-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.

The interplay between suckling, intestinal growth and brush-border membrane functions is critical during the perinatal period. The present study investigates changes in intestinal dimensions, activities of four brush border membrane hydrolases (lactase, sucrase, maltase and aminooligopeptidase) and rates of sugar and amino acid uptake by intact tissues and brush border membrane vesicles during the first 24 h of suckling. Total intestinal weight, mucosal weight and protein content increased 58%, 80% and 126% (P < 0.05) during the first 6 h of suckling; length and surface area did not increase. Total mucosal DNA content was 4.6-fold higher at 24 h after birth, with the rate of increase differing among intestinal regions. Hydrolytic capacities of the entire small intestine increased, more so for homogenates than for brush border membrane vesicles, and more for lactase relative to the other hydrolases studied. Rates of nutrient transport declined, especially for brush border membrane vesicles, for proximal and mid-intestine relative to distal intestine, and for glucose relative to galactose and amino acids. We conclude that 1) changes in brush border membrane digestive functions coincide with rapid intestinal growth, with postnatal patterns varying among hydrolases, transporters and regions; 2) insertion into the brush border membrane, not synthesis, limits the postnatal increase of hydrolase activity; and 3) despite declines in specific activity, hydrolytic and glucose transport capacities of the entire intestine remained stable or increased, and exceeded estimated dietary loads because of intestinal growth.

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.