2-Deoxyglucose transport and metabolism in Caco-2 cells

Abstract

We investigated the kinetics of 2-deoxy-D-glucose (DG) uptake and metabolism in Caco-2 cells, because this human cell line may represent a valid enterocyte model to assess the dynamics between sugar transport and metabolism and hence to obtain insights into the factors involved during the intracellular phase of glucose absorption. When studied in 14-day-old monolayers, DG uptake is characterized by a lag phase with a time course matching the decrease in intracellular glucose concentrations, 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 cells in substrate-free transport medium, however, steady-state DG uptake matches 2-deoxy-D-glucose 6-phosphate (DG-6-P) accumulation with undetectable levels of free DG. This complex behavior in DG uptake is linked to high hexokinase activity in Caco-2 cells, and the enzyme has a Michaelis-Menten constant K(m) for glucose that is typical of hexokinase 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 attributed 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 transport and hexokinase activity clearly shows that DG uptake is lowest in postconfluent cells when hexokinase is at its highest levels. We thus conclude that 1) transport is the rate-limiting step during DG accumulation, 2) G-6-P is a potent inhibitor of hexokinase activity compared with DG-6-P, so that enzyme inhibition may have physiological relevance in diverting glucose from metabolism during its active reabsorption in the small intestine, and 3) low levels of G-6-Pase activity seem to exclude this enzyme, and hence the endoplasmic reticulum, as important factors during the intracellular phase of glucose transport.