Carbon Metabolism of Enterobacterial Human Pathogens Growing in Epithelial Colorectal Adenocarcinoma (Caco-2) Cells

Analysis of the genome sequences of the major human bacterial pathogens has provided a large amount of information concerning their metabolic potential. However, our knowledge of the actual metabolic pathways and metabolite fluxes occurring in these pathogens under infection conditions is still limited. In this study, we analysed the intracellular carbon metabolism of enteroinvasive Escherichia coli (EIEC HN280 and EIEC 4608-58) and Salmonella enterica Serovar Typhimurium (Stm 14028) replicating in epithelial colorectal adenocarcinoma cells (Caco-2). To this aim, we supplied [U-13C6]glucose to Caco-2 cells infected with the bacterial strains or mutants thereof impaired in the uptake of glucose, mannose and/or glucose 6-phosphate. The 13C-isotopologue patterns of protein-derived amino acids from the bacteria and the host cells were then determined by mass spectrometry. The data showed that EIEC HN280 growing in the cytosol of the host cells, as well as Stm 14028 replicating in the Salmonella-containing vacuole (SCV) utilised glucose, but not glucose 6-phosphate, other phosphorylated carbohydrates, gluconate or fatty acids as major carbon substrates. EIEC 4608-58 used C3-compound(s) in addition to glucose as carbon source. The labelling patterns reflected strain-dependent carbon flux via glycolysis and/or the Entner-Doudoroff pathway, the pentose phosphate pathway, the TCA cycle and anapleurotic reactions between PEP and oxaloacetate. Mutants of all three strains impaired in the uptake of glucose switched to C3-substrate(s) accompanied by an increased uptake of amino acids (and possibly also other anabolic monomers) from the host cell. Surprisingly, the metabolism of the host cells, as judged by the efficiency of 13C-incorporation into host cell amino acids, was not significantly affected by the infection with either of these intracellular pathogens

In caco-2 cells, most of the "apical" SGLT1 resides in intracellular, microtubuli-associated vesicles

We investigated the distribution of the endogenous sodium/D-glucose cotransporter (SGLT1) in polarized Caco-2 cells, a model for enterocytes. A cellular organelle fraction was separated by free flow electrophoresis. As detected by an ELISA assay the major amount of SGLT1 resides in early endosomal fractions and only a minor amount in apical plasma membranes. The distribution-ratio between endosomes and apical membrane was approximately 2:1. Further immunochemical investigation of SGLT1 distribution by epifluorescence and confocal microscopy revealed that SGLT1 is located in small vesicles associated with microtubuli. Furthermore, the half-life of SGLT1 in Caco-2 cells was determined to be 2.5 d by metabolic labeling followed by immunoprecipitation. Since SGLT1 has a relatively long residence time, it is unlikely that intracellular SGLT1 populations are part of the synthesis/degradation pathway. We, therefore, propose that intracellular compartments containing SGLT1 are part of an endo-/exocytosis process which regulates SGLT1 abundance at the apical cell surface in response to altered physiological demands for D-glucose reabsorption. (This abstract is as it is printed in the journal)

Endosomal SGLT1 and its possible role in the regulation of D-glucose uptake

To elucidate the role of endosomal SGLTl in the regulation of sodium-dependent D-glucose uptake into enterocytes, we investigated the relationship between subcellular distribution of SGLTl and sodium-dependent 14C-methylglucose uptake into Caco-2 cells under varying conditions. Incubation with mastoparan shifted a large amount of SGLTl from the apical membrane to intracellular sites and significantly reduced sodiumdependent 14C-methylglucose uptake. We also investigated the effect of extracellular D-glucose levels. Cells pre-incubated with D-glucose free medium exhibited a significantly higher sodiumdependent 14C-methylglucose uptake than cells pre-incubated with high D-glucose medium. No difference in the overall subcellular distribution of SGLTl was observed between the two conditions. Interestingly, the change in 14C-methylglucose uptake was attenuated when microtubules were depolymerized. These results suggest that pharmacologically, D-glucose uptake can be regulated by a shift of the steady state distribution of carriers from the plasma membrane to endosomes. The physiological substrate D-glucose alters substrate uptake by an additional mechanism that requires microtubule-dependent vesicle transport without a change in the steady state distribution. This phenomenon could be explained by the hypothesis that SGLTl undergoes an activation/deactivation cycle during its constitutive cycling from endosomes to the plasma membrane and back

Endosomal SGLT1 and its possible role in the regulation of D-glucose uptake

To elucidate the role of endosomal SGLTl in the regulation of sodium-dependent D-glucose uptake into enterocytes, we investigated the relationship between subcellular distribution of SGLTl and sodium-dependent 14C-methylglucose uptake into Caco-2 cells under varying conditions. Incubation with mastoparan shifted a large amount of SGLTl from the apical membrane to intracellular sites and significantly reduced sodiumdependent 14C-methylglucose uptake. We also investigated the effect of extracellular D-glucose levels. Cells pre-incubated with D-glucose free medium exhibited a significantly higher sodiumdependent 14C-methylglucose uptake than cells pre-incubated with high D-glucose medium. No difference in the overall subcellular distribution of SGLTl was observed between the two conditions. Interestingly, the change in 14C-methylglucose uptake was attenuated when microtubules were depolymerized. These results suggest that pharmacologically, D-glucose uptake can be regulated by a shift of the steady state distribution of carriers from the plasma membrane to endosomes. The physiological substrate D-glucose alters substrate uptake by an additional mechanism that requires microtubule-dependent vesicle transport without a change in the steady state distribution. This phenomenon could be explained by the hypothesis that SGLTl undergoes an activation/deactivation cycle during its constitutive cycling from endosomes to the plasma membrane and back

Rôle de l' octapeptide KOGEOGPK issu du collagène de type III dans l' adhésion et la signalisation plaquettaires in vitro et dans la thrombose in vivo

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF