3-OMG Uptake Research Articles

Effects of valinomycin on hexose transport and cellular ATP pools in mouse fibroblasts.

The K+ ionophore valinomycin at concentrations of 1 X 10(-8) M and over, stimulated 2-deoxy-D-glucose (2DG) and 3-O-methylglucose (3OMG) uptake in Swiss 3T3 fibroblasts. The rate-limiting step of 2DG uptake was transport rather than phosphorylation, in the control or valinomycin-treated cells. Kinetic analysis showed that valinomycin increased the Vmax for 2DG uptake without change of the Km. The valinomycin-stimulated 2DG uptake was insensitive to 10 micrograms/ml cycloheximide, and extracellular K+ concentrations between 0.1 and 50 mM. On the other hand, valinomycin at the concentration of 1 X 10(-8) M and over, induced a rapid decrease in cellular ATP content, followed by stimulation of 2DG uptake and recovery of the ATP content. A similar relationship between the reduction of cellular ATP content and the subsequent stimulation of 2DG uptake was observed when the cells were treated not only with 2,4-dinitrophenol and iodoacetic acid, but also with other monovalent cation ionophores or inhibitors of oxidative phosphorylation. These results suggest that valinomycin may posttranslationally stimulate hexose transport by increasing the number of functional carriers of hexose or changing their mobility, and the rapid decrease in cellular ATP pools by valinomycin may be a trigger of the stimulation of the hexose transport in Swiss 3T3 fibroblasts.

Fluxes and compartmentation of 3-O-methyl-D-glucose in Riccia fluitans

In thalli of the aquatic liverwort, Riccia fluitans, 3-O-methyl-D-glucose (3-OMG) is not metabolized. Intracellular compartmentation, accumulation and transmembrane fluxes of 3-OMG have been determined by compartmental analysis. A novel set of equations has been derived to extend this method to non-steady-state conditions of constant but unequal unidirectional fluxes. Efflux kinetics with 3-OMG and L-glucose revealed two intracellular flux compartments, presumably cytoplasm and vacuole; an additional quickly exchanging compartment (half-time approx. 1 min) has been assigned to the apoplast. With 1 mM 3-OMG given externally, cytoplasmic 3-OMG concentration (c c) attains a quasi-steady state of about 10 mM lasting for >100 h, whereas the presumed vacuolar concentration (c v) rises steadily, but does not reach flux equilibrium even after two weeks (c v=46 mM). After 24 h incubation with 0.03 mM 3-OMG, c c=1 mM approx., and c v=3 mM approx., thus indicating accumulation by active hexose transport at both the plasmalemma and tonoplast. External D-glucose, but no D-mannitol, competitively inhibits 3-OMG uptake (cis-inhibition) and stimulates 3-OMG efflux at the plasmalemma by a factor up to 2.5. This trans-stimulation saturates half-maximally at 1.5 mM D-glucose. It clearly indicates a hexose carrier in the plasmalemma with one substrate-binding site for D-glocose and 3-OMG, alternately exposed to the cytoplasmic and outside compartment. The extent of the measured trans-stimulation can only be explained, if in the transport cycle the translocation of the empty substrate-binding site across the plasmalemma is rate-limiting.

Hexose transport and membrane depolarization in Riccia fluitans

In the aquatic liverwort Riccia fluitans, the uptake of (14)C-labeled 3-O-methyl glucose (3-OMG) and membrane depolarization (ΔΨ m ) caused by different hexoses has been studied as a function of time and concentration of hexose, K(+) and H(+), respectively. The rate of uptake of the non-metabolized 3-OMG shows two components: (A)A pH-dependent saturable uptake with a km value around 0.1 mM which saturates at 2.1 and 7.2 μmol G DW (-1) h(-1) at pH 6.8 and 5.0, respectively; and (B) a pH-insensitive uptake component which increases linearly with the external 3-OMG concentration and does not saturate ≦4 mM. Hexoses rapidly depolarize the plasmalemma of the thallus cell and increase its electrical conductance. The maximal ΔΨ m was 60±2 mV, the concentrations (mM) for half-maximal ΔΨ m were 0.24 glucose, 0.32 galactose, 0.37 2-deoxy glucose, 0.38 3-OMG, 0.57 mannose, and 34 fructose. In terms of a hexose carrier model and an equivalent circuit for the hexose-induced depolarized state of the membrane, it is proposed that a hexose carrier operates either electrogenically in its protonated, pH-and voltage-sensitive state, or by transmembrane diffusion of its uncharged state.

Membrane potentials and sugar transport by ATP-depleted intestinal cells: effect of anion gradients.

Rotenone or dinitrophenol treatment was used to decrease cellular ATP levels in isolated chicken intestinal epithelial cells by over 90% and to produce a cell population in which no steady-state accumulation of 3-O-methylglucose (3-OMG) against a concentration gradient is observed. In the presence of imposed inward-directed Na-anion gradients, these cells accumulate 3-OMG against a concentration gradient. The degree of maximal 3-OMG accumulation and initial influx stimulation in the presence of a given anion or combined Na-anion gradient can be correlated with the magnitude of the diffusion potential as determined by the membrane permeability of the given anion (SCN- > Cl- > isethionate- > SO42-). 3-OMG influx in the presence of a large NaCl gradient is comparable in ATP-depleted and normally energized cells. The slight difference in influx (energized > ATP depleted) is diminished by ouabain, suggesting that energized cells maintain a larger membrane potential (diffusion potential or rheogenic Na+-K+-ATPase ion pump-generated potential) than the ATP-depleted cells. Although initial rates of 3-OMG uptake into Na+-depleted normally energized cells also varies with the anion gradient, these differences disappear with time of incubation in Na+ or when cells are preincubated in Na+. In this situation, function of a rheogenic Na+ pump can establish a membrane potential in contrast to the case with ATP-depleted cells, which have a potential only as long as imposed ion gradients are maintained. All these experiments point to an important role for the electrical membrane potential as a driving force for Na+-dependent solute transport systems in both ATP-depleted and normally energized cells.

Time course of the effect of growth hormone in vitro on amino acid and monosaccharide transport and on protein synthesis in diaphragm of young normal rats.

The in vitro responsiveness of diaphragms from 18-day-old fasted normal rats to bovine growth hormone (bGH) with time was studied by determining the accumulation of a-aminoisobutyric acid (AIB) and 3–0 methylglucose (3-OMG) and by determining the incorporation of phenylalanine into diaphragm proteins. An estimate of the time course of the effect of GH was obtained by adding the different labeled substances to the incubation medium at different time periods after the start of the incubation. bGH (5 μg/ml) stimulated the uptake of AIB and 3-OMG and the incorporation of phenylalanine between 0–60 min after the start of the incubation. This period was then followed by a period of 60–120 min during which no further stimulation occurred in spite of the continuous presence of the hormone, i.e. the diaphragm was “refractory” to GH. When the incubation period was extended beyond the “refractory phase,” the uptake of AIB and the incorporation of phenylalanine once again increased in diaphragms exposed to bGH. The present experiments thus demonstrate that the membrane transport system (s) and protein synthesis in diaphragms of 18-day-old fasted rats pass through a sequence of changes in the response to GH. The fact that GH exerts a biphasic action on membrane transport and protein synthesis in diaphragm muscle of normal rats suggests that this responsiveness pattern to GH with time is a generalized cellular phenomenon in response to the hormone. (Endocrinology102: 1445, 1978)

2-Deoxyglucose transport by intestinal epithelial cells isolated from the chick.

Characteristics of 2-deoxyglucose uptake (2DG) by intestinal epithelial cells isolated from chickens were evaluated as a means of discriminating between the concentrative transport system for monosaccharides, associated with the mucosal brush border, and other possible routes of monosaccharide entry. 2DG was chosen as it is not a substrate for the mucosal transport system. The deoxysugar enters via a saturable pathway which is not Na+-dependent, is not inhibited by K+, does not accumulate solute against a concentration gradient; exhibits a high sensitivity to inhibition by phloretin; is relatively insensitive to phlorizin inhibition; and has low affinity [but high capacity relative to Na+-dependent mucosal transport of 3-O-methylglucose (3-OMG) and other monosaccharides]. These characteristics confirm those established in an earlier report for Na+-independent uptake of 3-OMG. Complications encountered in the use of 2DG as a test sugar include significant rates of metabolic conversion to an anionic form which presumably is a phosphorylated species. Methods for distinguishing between transport and subsequent metabolism are described. Inhibition of 2DG entry by several other sugars is described and inhibitory constants (K's) given for each.

Development of responsiveness of young normal rats to growth hormone

Growth hormone (GH) exerts a biphasic effect on the membrane transport of amino acids and sugars in diaphragms from hypophysectomized rats. A stimulatory (insulin-like) effect of GH is observed for approximately 3 hr after the administration of the hormone, and then the diaphragm becomes “refractory” to further administration of the hormone for 24–48 hr. In the present study the in vitro responsiveness of diaphragms from young normal rats of different ages to bovine growth hormone (bGH) was studied by measuring the accumulation of α-aminoisobutyric acid (AIB), cycloleucine, and 3-O-methylglucose (3-OMG) and by determining the incorporation of phenylalanine into diaphragm proteins. bGH increased the uptake of AIB in 6, 10, 14, 18, and 22-day old and of cycloleucine in 10, 14, and 18-day old rats, respectively. The maximal stimulatory effect was seen in 18-day old rats and no effects were seen in 26 and 30-day old animals. A similar pattern of age-related responsiveness was also seen when the uptake of 3-OMG and the incorporation of phenylalanine were studied. The rate of accumulation of the non-utilizable amino acids, as well as the rate of incorporation of phenylalanine, decreased markedly with age. Fasting the rats for 20 hr did not change the principal age-related responsiveness of AIB transport to GH, but the magnitude of the hormone effect increased due to a lower uptake of AIB in control diaphragms. bGH had a dose-dependent effect on the accumulation of AIB in fasted 18-day old rats, the threshold concentration of bGH being 0.1 μg/ml. When diaphragms from 18-day old rats were incubated with bGH for various periods, the uptake of AIB was stimulated between 0 and 60 and 120 and 180 min, but not between 60 and 120 min after the start of the incubation. This observation indicated that the muscles were “refractory” to GH between 60 and 120 min. Preincubation of diaphragms from 21-day old rats with bGH for 3 hr did not inhibit the stimulatory effect of bGH added subsequently indicating that the duration of the “refractory phase” did not exceed 270 min in this group of rats. When diaphragms from fasted rats of different ages were preincubated in Krebs bicarbonate buffer for 3 hr, a stimulatory effect of bGH on AIB uptake was also observed in 26 and 30-day old rats, possibly because the influence of endogenous GH subsided during the preincubation period. These results suggest that diaphragms from “old” normal rats are insensitive to exogenous GH because the “refractory phases” become considerably prolonged with age.