Deoxyglucose and 3‐O‐methylglucose transport in untreated and ATP‐depleted novikoff rat hepatoma cells. Analysis by a rapid kinetic technique, relationship to phosphorylation and effects of inhibitors

Abstract

Detailed time courses of uptake of labeled 3-O-methyl-D-glucose and 2-deoxy-D-glycose by untreated and ATP-depleted Novikoff rat hepatoma cells were determined as function of concentration (0.2-10 mM) by a rapid mixing/sampling technique which allows uptake measurements in time intervals as short as 1.5 seconds. Intracellular accumulation of 3-O-methylglucose in untreated and ATP-depleted cells and of deoxyglucose in ATP-depleted cells to equilibrium followed pseudo-first order kinetics and initial velocities were computed from overall time courses of substrate accumulation. Initial velocity was a Michaelis-Menten function of exogenous substrate concentration. The estimated kinetic constants for zero-trans transport of 3-O-methylglucose were about the same for untreated and ATP-depleted cells (Kztm = 1.73 +/- 0.24 mM; Vztmax = 28.8 +/- 3.6 pmoles/microliter cell H2O. sec) and were similar to those for deoxyglucose transport in ATP-depleted cells (Kztm = 0.65 +/- 0.1 mM; Vztmax = 19.6 +/- 1.6 pmoles/microliter cell H2O. sec). Similar kinetic parameters were obtained for the transport of D-glucose and D-galactose in ATP-depleted cells. The transport of 3-O-methylglucose and deoxyglucose were inhibited by each other in a simple competitive manner with apparent Ki's similar to their transport Km's. In untreated cells, in which deoxyglucose was phosphorylated, intracellular steady-state levels of free deoxyglucose accumulated within 10 to 20 seconds of incubation regardless of its concentration in the medium. Thereafter, the rate of deoxyglucose incorporation into total cell material reflected the rate of phosphorylation rather than the transport rate. The rate of deoxyglucose transport exceeded the initial rate of its phosphorylation by 20-40 %. The intracellular steady-state-levels observed during the first 2 minutes of incubation decreased from about 40% of equilibrium level at 0.2 mM deoxyglucose to about 8% at 10 mM. Computer fits of a kinetic equation describing transport and phosphorylation as independent processes operating in tandem to these data are consistent with the observed kinetic constants for hexose transport and hexokinase activity with deoxyglucose as substrate. Upon longer incubation (2-10 minutes) the rate of deoxyglucose uptake by the phosphorylating cells decreased progressively, concomitant with a decrease in intracellular ATP and an increase in intracellular deoxyglucose to equilibrium levels. It is demonstrated that the rate of deoxyglucose uptake, measured at two or more minutes, seriously underestimates the hexose transport rate and yields misleading conclusions regarding the extent and type of inhibition by transport inhibitors, such as persantin or cytochalasin B. Persantin inhibited hexose transport in a simple non-competitive manner (Ki = 20 muM) indicating that the drug affects the function of the hexose carrier.

Acute regulation of glucose transport in a monocyte–macrophage cell line: Glut-3 affinity for glucose is enhanced during the respiratory burst

“…Hence the differences between 2-DOG and 3-O-MG uptake and K m could be explained by the phosphorylation of 2-DOG by hexokinase. Higher K m values were observed when 3-O-MG was used ; this has been reported by other investigators using other cell types [32][33][34][35][36]. Differences in uptake between the two hexoses were also observed and these would have contributed to the differences in V max .…”

Hexose transport in L6 rat myoblasts. I. Rate‐limiting step, kinetic properties, and evidence for two systems

“…Similar findings have been observed in rat adipocytes (Olefsky, 1978). On the other hand, mouse 3T3 cells and Novikoff rat hepatoma cells are found to contain significant steady-state levels of free intracellular 2-DOG at early times during uptake, indicating that phosphorylation, not transport, is the rate-limiting step of 2-DOG uptake (Colby and Romano, 1975;Romano, 1976;Graff et al, 1978).…”

“…Recent studies indicate that hexose transport is the rate-limiting step in rat adipocytes (Olefsky, 1978) and L6 myoblasts (Lo and Duronio, 1984a;Klip et al, 1982). In mouse 3T3 cells and Novikoff rat hepatoma cells, though, phosphorylation was shown to be the rate-limiting step (Romano, 1976;Graff et al, 1978). In this article we provide a detailed investigation to demonstrate that transport is the rate-limiting step of hexose uptake into L6 cells.…”

Modulation of the hypoxic toxicity and binding of misonidazole by glucose

“…The rate of HMP was a measure of the extent of redutive metabolism in the cells. The HMP had been shown to increase in the presence of MISO (Varnes et al, 1984 (Graff et al, 1978;Weber, 1973), which had been shown to be selective and phloretinsensitive (Ling & Sutherland, 1986a). As shown in Figure 1, the initial rate of this transport in EMT6/Ro cells was also directly dependent on the extracellular concentration up to 5 mm glucose.…”