During intestinal inflammation TNF¿ levels are increased and as a consequence malabsorption of nutrients may occur. We have previously demonstrated that TNF¿ inhibits galactose, fructose and leucine intestinal absorption in animal models. In continuation with our work, the purpose of the present study was to investigate in the human intestinal epithelial cell line Caco-2, the effect of TNF¿ on sugar transport and to identify the intracellular mechanisms involved.
Caco-2 cells were grown on culture plates and pre-incubated during different periods with various TNF¿ concentrations before measuring the apical uptake of galactose, ¿-methyl-glucoside (MG) or fructose for 15min. To elucidate the signaling pathway implicated, cells were pre-incubated for 30min with the PKA inhibitor H-89 or the PKC inhibitor chelerythrine, before measuring the sugar uptake. The expression in the apical membrane of the transporters implicated in the sugars uptake process (SGLT1 and GLUT5) was determined by Western blot.
TNF¿ inhibited 0.1mM MG uptake after pre-incubation of the cells for 6-48h with the cytokine and in the absence of cytokine pre-incubation. In contrast, 5mM fructose uptake was stimulated by TNF¿ only after long pre-incubation times (24 and 48h). These effects were mediated by the binding of the cytokine to its specific receptor TNFR1, present in the apical membrane of the Caco-2 cells. Analysis of the expression of the MG and fructose transporters at the brush