2-DOG Uptake Research Articles

Functional heterogeneity of human cord blood monocytes.

Human cord blood monocytes were separated into four different subpopulations by means of a discontinuous bovine serum albumin (BSA) gradient. Of the least dense, 31% were present in fraction A, 17% in fraction B and 13% in fraction C and of the most dense, 20% were in fraction D. The rest (17%) sedimented as a pellet, of which 93% were dead cells. The monocytes of fraction C (density greater than or equal to 1.070) demonstrated suppressor activity on in vitro antibody synthesis of maternal B cells. Fraction D (density greater than or equal to 1.075) monocytes enhanced antibody synthesis of maternal B cells compared with synthesis produced in a similar experiment with unfractionated monocytes. Addition of either fraction A or B monocytes to the mixed culture of T and B cells resulted in antibody production comparable to that produced by addition of unfractionated monocytes. The functional heterogeneity of the cord monocytes was assayed also by 2-deoxyglucose (2-DOG) uptake, a marker for immunological macrophage activation. Fractions A and D showed significantly higher 2-DOG transport than that of unfractionated monocytes; in contrast, fraction C showed a 50% reduction of 2-DOG uptake. Furthermore, in contrast to fraction D, fraction C possessed only minimal phagocytic activity (for antibody-coated sheep erythrocytes) and minimal hemoxygenase enzyme activity. These data demonstrate functional heterogeneity of cord blood monocytes.

12-0-tetradecanoylphorbol-13-acetate and concanavalin A enhance glucose uptake in thymocytes by different mechanisms.

The effects of the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) and the plant lectin concanavalin A (Con A) on glucose uptake in murine thymocytes were studied. TPA induces a rapid dose-dependent increase in the uptake of 2-deoxyglucose and in the transport of 3-0-methylglucose. Con A also elicits a time- and dose-dependent enhancement of 2-deoxyglucose uptake. The effect of Con A, however, is less pronounced. The effect of combined treatment of thymocytes with Con A and TPA is not additive. Cytochalasin B completely inhibits the basal, as well as TPA- and Con A-enhanced, 2-deoxyglucose uptake. Dexamethasone markedly inhibits basal 2-deoxyglucose uptake, but is less inhibitory to enhanced 2-deoxyglucose uptake induced by TPA and Con A. The effect of TPA on 2-deoxyglucose uptake and 3-0-methylglucose transport is refractory to inhibition by isobutyl methyl xanthine, dibutyryl cyclic AMP, and ethyleneglycol tetraacetic acid. These agents markedly inhibit the enhancement of 2-deoxyglucose (2-DOG) uptake by Con A. p-Bromophenacyl bromide, an inhibitor of phospholipase A2, also selectively inhibits Con A enhancement of 2-DOG uptake. Taken together, the results suggest that Con A and TPA exert their stimulatory effect on glucose uptake by different activating mechanisms, but they may share a final common transport pathway.

Stimulation of sugar transport in cultured heart cells by triiodothyronine (T3) covalently bound to red blood cells and by T3 in the presence of serum.

T3, covalently bound to red blood cells (RBCs), stimulated the uptake rate of 2-deoxy-D-glucose (2-DOG) in cultured chick embryo heart cells. The response, measured 6 h after exposure, was at least the same than that to free T3. An inhibitor of rhodamine-T3 internalization, bacitracin, did not affect the stimulation of sugar uptake by T3 regardless of whether T3 was covalently bound or free. Pretreatment of RBC-T3 with anti-T3 immunoglobulin G completely blocked the effect of T3, whereas normal rabbit immunoglobulin G failed to do so. Addition of 5% chick serum to the medium stimulated 2-DOG uptake to 144% of the control at 6 h. Adding T3 (10 nM) to the serum-containing medium increased 2-DOG uptake to 171% of the control. The effect of T3 alone or in the presence of serum was not inhibited by cycloheximide, puromycin, or actinomycin D. A T3 dose response curve, in medium containing 10% dehormonized serum, showed enhancement of the T3 effect when compared with the curve obtained in the serum-free medium. The minimal effective concentration of T3 was 10 pM in the presence of serum and 100 pM in its absence. The slope of the linear portion of the dose response curve was greatly increased and the maximal response markedly enhanced by serum. The ED50 was 0.33 nM vs. 0.43 nM in terms of total T3 concentration and 0.16 nM vs. 0.43 nM in terms of free T3 in the presence or absence of serum, respectively. These data suggest that T3, in physiological concentrations, activates sugar transport through an external contact with the cell surface.

Neuronal cues regulate uptake in cultured astrocytes

Astrocytes of the vertebrate central nervous system are postulated to nourish neurons4,~0,13, to electrically insulate cortical neuronal columns 15, to cue patterns of developmental migrations of neurons 14, and to regulate the ionic environment of brain 5, 17,~9. Despite persistent investigation, the function or functions of this phylogenetically ubiquitous cell type remain largely hypothetical. Neurons and their attendant glia are considered to be a functional metabolic unit. For this to be the case, at least two criteria must be met: (1) some mechanism must exist for communication of relevant information about the metabolic state of one cell-type to the other; and (2) either cell-type must be able to alter its metabolism in response to cues emitted by the other. Although the spectrum of neuronally emitted cues is large, ideally, such cues should reflect some aspect of neuronal activity. In the present study we have selected extracellular K + and 3 putative neurotransmitters as neuronal cues, since it is known that the cerebral levels of both reflect neuronal activity and that each also affects glial membrane potentiala, 18, oxygen uptake 1 and cyclic nucleotide levels 3,9,11. One manner in which neurons may affect glial metabolism is by inducing alterations in the glial uptake of key metabolites, such as glucose, possibly a rate-limiting step in cerebral glycolysis 16 and selected amino acids, the uptake of which by the glial cells may, in part, reflect transfer from the extracellular space of brain tissue 7,12. Recently, we developed a technique for the cultivation of a purified population of astrocytes, derived from neonatal rat forebrain 2 and in the present study we investigate the effect of neuronal cues (see above) on the astrocytic uptake of the glucose analogue 2-deoxy-D-glucose (2-DOG) and of L-methionine (MET), the precursor amino acid essential for the cerebral synthesis of the universal methyl donor molecule, S-adenosylL-methionine. Under the conditions of the experiment described in Fig. 1, the uptake of 2-DOG remained linear for at least 15 min. To determine the effects of extracellular K + on the uptake of 2-DOG, KC1 replaced NaCI on a molar basis. Plates of confluent cells were incubated with 0.25, 0.50, and 1.0 mM 2-DOG at the indicated concentrations of extra-

The effects of 1,7-bis( p-aminophenoxy)heptane (153C51) on glucose utilization by Schistosoma mansoni

1. There was no consistent change in glycogenolysis in S. mansoni males and females incubated together in vitro 24 hr after treatment of the host with a therapeutic dose of 153C51. 2. In similar incubations, glycolysis was significantly reduced only when the lowest concentration of glucose (0.4 mM) was added to the medium. 3. Experiments on the uptake of 0.5 mM D-[U- 14C] glucose and the non-metabolized analogue 2-deoxy- D-[1- 3H] glucose by pairs and by male and female schistosomes incubated separately indicated that the inhibition of utilization of glucose from low substrate concentration in males and females incubated together could be explained by an inhibition of glucose uptake in male worms. Uptake by female worms was unaffected by drug treatment. 4. The rapid accumulation of radioactivity derived from 1.7-bis( p-aminophenoxy)[1.7- 14C]heptane preceded the onset of inhibition of 2DOG uptake by male parasites by several hours. Throughout the period 1–24 hr after treatment of the host, female schistosomes were approximately 50% more radioactive than males yet in this study they were almost totally unaffected by the drug. 5. The accumulation by schistosomes of radioactivity derived from 153C51 labelled in different positions accords with earlier work on the basic chemical structure essential for schistosomicidal activity in this type of compound. 6. The similarity in the glycogen reserves of pairs of control parasites and those pairs removed from treated mice together with the results of the experiments on glucose utilization in vitro make it unlikely that the interference with glucose uptake observed in vitro in male parasites could be a primary cause of schistosome death or even of physiological distress in 153C51-treated parasites in vivo.

Membrane transport by guinea pig peritoneal exudate leukocytes: effect of phagocytosis on hexose and amino acid transport.

Short term, carrier mediated transport of D-glucose, L-leucine and L-lysine by guinea pig peritoneal macrophages was characterized. Analysis of the amino acid transport demonstrated two-limbed double reciprocal plots suggesting two transport systems for each amino acid. The low concentration limb of the curves established a Km of 0.1 mM for L-leucine and 0.05 mM for L-lysine; Vmax values were 2.0 and 2.85 nmole/mg protein/90 seconds, respectively. Leucine and lysine were shown to be competitive inhibitors of each other. Further competition studies revealed that other amino acids also had affinity for these carriers. Amino acid transport was found to be sensitive to sulfhydryl active compounds. Colchicine treatment of peritoneal macrophages did not inhibit the transport of the amino acids tested. Preloading macrophages with latex beads or heat-killed staphylococci by phagocytosis stimulated 2-deoxy-D-glucose (2-dOG) uptake markedly, but had no measurable effect on amino acid transport. Although total transport of 2-dOG increased in post-phagocytic macrophages, the kinetics of the system were not altered significantly. The Km for both pre- and post-phagocytic transport of 2-dOG was shown to be 1.2 mM and the Vmax was shown to increase from a pre-phagocytic value of 20 nmoles/mg protein/90 seconds to a post-phagocytic 27 nmoles/mg protein/90 seconds. Phagocytosis of heat-killed staphylococci by guinea pig polymorphonuclear leukocytes (PMNs), however, did not cause an augmentation in hexose transport in the cells. The presence of colchicine during phagocytosis did not alter subsequent uptake of amino acids by the macrophages.

Is glucose transport enhanced in virus‐transformed mammalian cells? A dissenting view

Much of the literature on the uptake of glucose by untransformed and transformed animal cells is based on experiments carried out with 2-deoxy-D-glucose (2-DOG). Results obtained with this analog can be ambiguous, since 2-DOG can be phosphorylated by hexokinases of animal cells. An intracellular trapping mechanism is thus provided. Therefore, the total flux of 2-DOG into the cell is a resultant of both transport and hexokinase action, and the measurement of total 2-DOG incorporation is a valid measurement of transport only if 2-DOG is phosphorylated as rapidly as it enters the cell. Evidence is presented here that this is not necessarily the case, significant levels of free intracellular 2-DOG approaching external concentrations were found in untransformed and transformed mouse 3T3 cells even at early times during uptake. Differences in total intracellular 2-DOG between untransformed and transformed cells were accounted for entirely by 2-deoxyglucose phosphate. Thus, it appears the apparent increase of 2-DOG uptake accompanying transformation in these cell lines is not due to an effect on the transport process, but on enhanced phosphorylation, which is a reflection of an alteration in the regulation of glycolysis. The ambiguity introduced by phosphorylation can be oviated by the use of an analog that cannot be phosphorylated, such as 3-O-methyl-D-glucose. The rate of transport and efflux of this sugar was not found to be different in untransformed versus transformed 3T3 cells. Moreover, deficiencies of this analog as a substrate for the glucose transport system are pointed out.

Decreased basal, noninsulin-stimulated glucose uptake and metabolism by skeletal soleus muscle isolated from obese-hyperglycemic (ob/ob) mice.

Insulin resistance of diaphragms of ob/ob mice has been repeatedly demonstrated previously both in vitro and in vivo. In the present study, transport and metabolism of glucose with and without insulin stimulation were compared in a skeletal muscle more likely than diaphragm or heart to be representative of the overall striated muscle mass, i.e. isolated soleus muscle. Compared with soleus muscle from lean controls, unstimulated lactate release in the presence of exogenous glucose was depressed from 16.2 to 12.3 nmol/60 min per mg wet wt in soleus from ob/ob mutants; glycolysis was decreased from 6.6 to 3.7 and [14C]glucose oxidation to 14CO2 from 0.90 to 0.33 nmol glucose/60 min per mg wet wt. Uptake of 2-deoxyglucose (2-DOG), both with and without insulin, was very much less for soleus from ob/ob than from lean mice, at 2-DOG concentrations ranging from 0.1 to 10 mM, and in mice of 6-15 wk. When 2-DOG concentration was 1 mM, its basal uptake was 0.53 nmol/30 min per mg wet wt for soleus of ob/ob as against 0.96 for soleus of lean mice. The absolute increment due to 1 mU/ml insulin was 0.49 in muscle of ob/ob as against 1.21 in that of lean mice. When the resistance to insulin action was decreased by pretreatment in vivo by either streptozotocin injection or fasting, the decreased basal 2-DOG uptake of subsequently isolated soleus muscle was not improved. Inhibition of endogenous oxidation of fatty acids by 2-bromostearate, while greatly increasing 14CO2 production from [14C]glucose, did not affect basal [5-3H]glucose metabolism or 2-DOG uptake. It is suggested that transport and/or phosphorylation of glucose under basal, unstimulated conditions are depressed in soleus muscle of ob/ob mice, whether or not resistance to insulin and hyperinsulinemia are also present. Although the origin of the decreased basal glucose uptake remains unknown it might be related to a similar decrease in basal glucose uptake by ventromedial hypothalamic cells, an event presumably resulting in a tendency to hyperphagia. Decreased basal glucose uptake by soleus muscle of ob/ob mice might explain the hyperglycemia, and hence partly the hyperinsulinemia and excessive fat deposition of those animals.

Specificity of the glucose transport system in Leishmania tropica promastigotes.

The glucose transport system in Leishmania tropica promastigotes was characterized by the use of labeled 2-deoxy-D-glucose (2-DOG), a nonmetabolizable glucose analog. The uptake system has a Q10 of 2 and a heat of activation of 10.2 kcal/mole. The glucose transport system is subject to competitive inhibition by 2-DOG, glucosamine, N-acetyl glucosamine, mannose, galactose, and fructose which suggests that substitutions in the hexose chain at carbons 2 and 4 do not affect carrier specificity. In contrast, changes at carbon 1 (alpha-methyl-D-glucoside, 1,5-anhydroglucitol) and carbon 3 (3-0-methyl glucose) lead to loss of carrier affinity since these sugars do not compete for the glucose carrier. Sugars that compete with the glucose carrier have one common feature--they all exist in the pyranose form in solution. The carrier for D-glucose does not interact with L-glucose or any of the pentose sugars tested. Uptake of 2-DOG is inhibited by glycerol. This inhibition, however, is noncompetitive; it is evident; therefore, that glucose and glycerol do not compete for the same carrier. Glycerol does not repress the glucose carrier since cells grown in presence of glycerol transport the sugar normally.

Phosphorylation but not transport of sugars is enhanced in virus‐transformed mouse 3T3 cells

The transport and phosphorylation of 2-deoxy-D-glucose are separate and sequential events in both normal and virus-transformed 3T3 cells. The apparent enhancement of 2-dOG uptake by 3T3 cells accompanying virus transformation is not due to an effect on the transport process but to enhanced phosphorylation by intracellular kinases. Phosphorylation of 3-O-methyl-D-glucose does not occur in these cells. Both the rate and extent of transport of this glucose analog is the same in normal cells, SV40 virus-transformed cells and sarcoma virus-transformed cells. The appropriateness of using 3-O-MeG for studies of the glucose transport system of animal cells is examined and discussed.