2-DEOXYGLUCOSE TRANSPORT AND METABOLISM IN CACO-2 CELLS

We investigated the kinetics of 2-deoxy-D-glucose (DG) uptake and meta bolism in Caco-2 cells, because this human cell line may represent a v alid enterocyte model to assess the dynamics between sugar transport a nd metabolism and hence to obtain insights into the factors involved d uring the intracellular phase of glucose absorption. When studied in 1 4-day-old monolayers, DG uptake is characterized by a lag phase with a time course matching the decrease in intracellular glucose concentrat ions, and no intracellular glucose 6-phosphate (G-6-P) can be detected at any time during incubation. After 1 h of preincubation of Caco-2 c ells in substrate-free transport medium, however, steady-state DG upta ke matches 2-deoxy-D-glucose B-phosphate (DG-B-P) accumulation with un detectable levels of free DG. This complex behavior in DG uptake is li nked to high hexokinase activity in Caco-2 cells, and the enzyme has a Michaelis-Menten constant (K-m) for glucose that is typical of hexoki nase type II (0.120 +/- 0.003 mM). Caco-2 cells also contain low-level glucose-6-phosphatase (G-6-Pase) activity, which may account for the leveling off in DG uptake, and the kinetics of DG transport may be att ributed to the existence of a predominant pathway with a K-m of 1.7 +/ - 0.2 mM. Finally, analysis of the growth-related expression of DG tra nsport and hexokinase activity clearly shows that DG uptake is lowest in postconfluent cells when hexokinase is at its highest levels. We th us conclude that I)transport is the rate-limiting step during DG accum ulation, 2) G-6-P is a potent inhibitor of hexokinase activity compare d with DG-6-P, so that enzyme inhibition may have physiological releva nce in diverting glucose from metabolism during its active reabsorptio n in the small intestine, and 3) low levels of G-6-Pase activity seem to exclude this enzyme, and hence the endoplasmic reticulum, as import ant factors during the intracellular phase of glucose transport.