Effect of K+ channel-blockers on sugar uptake by isolated chicken enterocytes

The effects of Ba2+, quinine, verapamil, and Ca2(+)-free saline solutions on sugar active transport have been investigated in isolated chicken enterocytes. Ba2+, quinine, and verapamil, which have been shown to inhibit Ca2(+)-activated K+ channels, decreased basal and theophylline-dependent 3-O-methylglucose (3-O-MG) accumulation. Ca2(+)-free conditions reduced 3-O-MG uptake in theophylline-treated enterocytes, but it had no effect in control cells. On the other hand, the uptake of a non-actively transported sugar, 2-deoxyglucose (2-DOG), by control or theophylline-treated cells was not modified by the presence of verapamil or by Ca2(+)-removal. 3-O-MG increased ouabain-sensitive Na(+)-efflux, but had no effect on either K+ efflux or K+ uptake. However, in the presence of Ba2+, K+ uptake was stimulated by 3-O-MG, and this increase was prevented by ouabain. All these findings are discussed in terms of the role that K+ permeability may play in cellular homeostasis during sugar active transport.     

Transport of sugars in chick-embryo fibroblasts. Evidence for a low-affinity system and a high-affinity system for glucose transport.

The rate of D-glucose uptake by cells that had been deprived of sugar for 18-24h was consistently observed to be 15-20 times higher than that in control cells maintained for the same length of time in medium containing glucose. This increased rate of glucose transport by sugar-starved cells was due to a 3-5-fold increase in the Vmax. value of a low-affinity system (Km 1 mM) combined with an increase in the Vmax of a separate high-affinity system (Km 0.05-0.2 mM). The high-affinity system, which was most characteristic of starved cells, was particularly sensitive to low concentrations of the thiol reagent N-ethylmaleimide; 50% inhibition of uptake occurred at approx. 0.01 mM-N-ethylmaleimide. In contrast with the high-affinity system, the low-affinity system of either the fed cells or the starved cells was unaffected by N-ethylmaleimide. In addition to the increases in the rate of D-glucose transport, cells deprived of sugar had increased rates of transport of 3-O-methyl-D-glucose and 2-deoxy-D-glucose. No measurable high-affinity transport system could be demonstrated for the transport of 3-O-methylgucose, and N-ethylmaleimide did not alter the initial rate. Thus the transport of 3-O-methyglucose by both fed and starved cells was exclusively by the N-ethylmaleimide-insensitive low-affinity system. The low-affinity system also appeared to be the primary means for the transport of 2-deoxyglucose by fed and starved cells. However, some of the transport of 2-deoxyglucose by starved cells was inhibited by N-ethylmaleimide, suggesting that 2-deoxyglucose may also be transported by a high-affinity system. The results of experiments that measured transport kinetics strongly suggest that glucose can be transported by a least two separate systems, and 3-O-methylglucose and 2-deoxyglucose by one. Support for these interpretations comes from the analysis of the effects of N-ethylmaleimide and cycloheximide as well as from the results of competition experiments. The uptake of glucose is quite different from that of 2-deoxyglucose and 3-O-methylglucose. The net result of sugar starvation serves to emphasize these differences. The apparent de-repression of the transport systems studied presents an interesting basis for further studies of the regulation of transport in a variety of cells.     

Transport and phosphorylation of hexoses in normal and rous sarcoma virus-transformed chick embryo fibroblasts

Effects of transformation by Rous sarcoma virus on sugar uptake and activity and the subcellular distribution of hexokinase isozymes in chick embryo fibroblasts were examined. Transformation caused a several-fold increase in the maximum velocity for uptake of 2-deoxyglucose without a significant change in K_m. Cytochalasin B (CB), was used to differentiate between the effects of transformation on facilitated diffusion and the nonsaturable (CB-insensitive) mode. Transformation was found to stimulate 2-deoxyglucose transport by both mechanisms, but the increase in transport by the CB-insensitive mode was greater. Transformation enhances the activity of hexokinase, the enhancement being confined to the particulate fraction of the enzyme. Heat-inactivation and electrophoretic mobility studies showed that although hexokinase Type I is the major form in both normal and transformed fibroblasts, there is a significant increase in the proportion of the Type II isozyme in the transformed cells.     

Hexose and amino acid transport by chicken embryo fibroblasts infected with temperature-sensitive mutant of Rous sarcoma virus. Comparison of transport properties of whole cells and membrane vesicles

The effect of transformation on hexose and amino acid transport has been studied using whole cells and membrane vesicles of chicken embryo fibroblasts infected with the temperature-sensitive mutant of the Rous sarcoma virus, TS-68. In whole cells, TS-68-infected chicken embryo fibroblasts cultured at the permissive temperature (37 degrees C) had a 2-fold higher rate of 2-deoxy-D-glucose uptake than the same cells cultured at the non-permissive temperature (41 degrees C). However, both the non-transformed and transformed cells had comparable rates of alpha-aminoisobutyric acid transport. Membrane vesicles, isolated from TS-68-infected chicken embryo fibroblasts cultured at 41 degrees C or 37 degrees C, displayed carrier-mediated, intravesicular uptake of D-glucose and alpha-aminoisobutyric acid. Membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 37 degrees C had an approx. 50% greater initial rate of stereospecific hexose uptake than the membrane vesicles from fibroblasts cultured at 41 degrees C. The two types of membrane vesicle had similar uptake rates of alpha-aminoisobutyric acid. The results of hexose and amino acid uptake by the membrane vesicles correlated well with those observed with the whole cells. Km values for stereospecific D-glucose uptake by the membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 41 and 37 degrees C were similar, but the V value was greater for the membrane vesicles from TS-68-infected cells cultured at 37 degrees C. Cytochalasin B competitively inhibited stereospecific hexose uptake in both types of membrane vesicle. These findings suggest that the membrane vesicles retained many of the features of hexose and amino acid transport observed in whole cells, and that the increased rate of hexose transport seen in the virally-transformed chicken embryo fibroblasts was due to an increase in the number or availability of hexose carriers.     

Hexose and amino acid transport by chicken embryo fibroblasts infected with temperature-sensitive mutant of rous sarcoma virus: Comparison of transport properties of whole cells and membrane vesicles

The effect of transformation on hexose and amino acid transport has been studied using whole cells and membrane vesicles of chicken embryo fibroblasts infected with the temperature-sensitive mutant of the Rous sarcoma virus, TS-68. In whole cells, TS-68-infected chicken embryo fibroblasts cultured at the permissive temperature (37°C) had a 2-fold higher rate of 2-deoxy-d-glucose uptake than the same cells cultured at the non-permissive temperature (41°C). However, both the non-transformed and transformed cells had comparable rates of α-aminoisobutyric acid transport. Membrane vesicles, isolated from TS-68-infected chicken embryo fibroblasts cultured at 41°C or 37°C, displayed carrier-mediated, intravesicular uptake of d-glucose and α-aminoisobutyric acid. Membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 37°C had an approx. 50% greater initial rate of stereospecific hexose uptake than the membrane vesicles from fibroblasts cultured at 41°C. The two types of membrane vesicle had similar uptake rates of α-aminoisobutyric acid. The results of hexose and amino acid uptake by the membrane vesicles correlated well with those observed with the whole cells. K_m values for stereospecific d-glucose uptake by the membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 41 and 37°C were similar, but the V value was greater for the membrane vesicles from TS-68-infected cells cultured at 37°C. Cytochalasin B competitively inhibited stereospecific hexose uptake in both types of membrane vesicle. These findings suggest that the membrane vesicles retained many of the features of hexose and amino acid transport observed in whole cells, and that the increased rate of hexose transport seen in the virallytransformed chicken embryo fibroblasts was due to an increase in the number or availability of hexose carriers.     

Hexose uptake enhancing factor released from rous sarcoma cells

Conditioned media from Rous sarcoma virus transformed chicken embryo fibroblasts stimulate the uptake of 2-deoxyglucose in normal chicken fibroblasts. The factor responsible for this effect, which is also shed in very low amount by non-transformed fibroblasts, is destroyed by trypsin and not linked to the protease and plasminogen activator activities present in the media. Its apparent molecular weight, determined by gel filtration, is about 20.000 daltons. The factor released by transformed cells might be related to the monomeric form of a family of glucose binding and transport proteins recently reported by Lee and Lipmann ('78) to be detached by detergents from normal and transformed cells.     

Hydrolase and serum treatment of normal chick embryo cells: effects on hexose transport.

We have asked whether treatment of normal cultured cells with proteases, other hydrolytic enzymes, or serum can convert them into transient phenocopies of transformed cells with respect to the very high rate of hexose transport characteristic of transformed cells. Treatment of density-inhibited cultures of normal chick embryo fibroblasts with trypsin, plasmin, neuraminidase, or hyaluronidase stimulated their rate of 2-deoxyglucose uptake to a level only marginally higher than that seen in normal exponentially growing cultures, and only 35-45% of that seen in transformed cultures. Addition of the hydrolytic enzymes to growing cell cultures had little effect on 2-deoxyglucose uptake. Serum, however, could stimulate 2-deoxyglucose uptake all the way up to the transformed level. Even though the hydrolases and serum differed in their ability to stimulate 2-deoxyglucose uptake, both reagents were capable of stimulating cell division equally well. Evidence is presented suggesting that the hexose transport rate is controlled by serum factors, and that proteolysis can affect the response of the cells of these factors.     

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.     

High glucose concentrations inhibit glucose phosphorylation, but not glucose transport, in human endothelial cells.

Glucose uptake is autoregulated in a variety of cell types and it is thought that glucose transport is the major step that is subjected to control by sugar availability. Here, we examined the effect of high glucose concentrations on the rate of glucose uptake by human ECV-304 umbilical vein-derived endothelial cells. A rise in the glucose concentration in the medium led a dose-dependent decrease in the rate of 2-deoxyglucose uptake. The effect of high glucose was independent of protein synthesis and the time-course analysis indicated that it was relatively slow. The effect was not due to inhibition of glucose transport since neither the expression nor the subcellular distribution of the major glucose transporter GLUT1, nor the rate of 3-O-methylglucose uptake was affected. The total in vitro assayed hexokinase activity and the expression of hexokinase-I were similar in cells treated or not with high concentrations of glucose. In contrast, exposure of cells to a high glucose concentration caused a marked decrease in phosphorylated 2-deoxyglucose/free 2-deoxyglucose ratio. This suggests the existence of alterations in the rate of in vivo glucose phosphorylation in response to high glucose. In summary, we conclude that ECV304 human endothelial cells reduce glucose utilization in response to enhanced levels of glucose in the medium by inhibiting the rate of glucose phosphorylation, rather than by blocking glucose transport. This suggests a novel metabolic effect of high glucose on cellular glucose utilization.     

Loss of the post-translational control of nutrient transport in vitro and in vivo virus-transformed chicken cells.

The removal of serum from the medium of uninfected fibroblasts decreased the rate of uptake of uridine, 2-deoxyglucose, alpha-aminoisobutyrate and thymidine. Its subsequent addition rapidly and reversibly stimulated the uptake of all the nutrients but thymidine and this response was not inhibited by treatment of the cells with cycloheximide. The cycloheximide insensitive, rapid increase in the rate of transport has been designated post-translational control. The nutrient transport systems in chick embryo fibroblasts transformed in vitro with avian sarcoma viruses and virus-induced cultured chicken tumor cells do not respond to serum removal or addition. Two possible levels for the control of nutrient transport, i.e., mitogen receptor occupancy and mitrogen-induced activation of the transport system, are presented to explain these observations.     

The pH-dependence of sugar-transport and glycolysis in cultured Ehrlich ascites-tumour cells.

1. pH-dependence of glycolysis has generally been ascribed to the effects of pH on the activities of glycolytic enzymes. The present study shows that sugar transport is pH-dependent in cultured Ehrlich ascites-tumour cells. 2. The rates of glucose consumption, of 3-O-methylglucose transport, and of 2-deoxyglucose transport and phosphorylation increased as linear functions of pH, as the pH of the cell culture medium was increased from 6.1 to 8.5. Transport of glucose, as measured in ATP-depleted cells, was pH-dependent to the same extent as transport of the non-metabolizable sugars. 3. Glucose consumption rates were about 8-fold higher at pH 8.5 than at pH 6.4. About 65-85% of glucose was converted into lactate. Sugar transport rates were 2.5-fold higher at pH 8.5 than at pH 6.3. 4. pH affected both simple diffusion and facilitated diffusion. pH effect was mainly on the Vmax. of 2-deoxyglucose uptake, and on the rapid-uptake phase of 3-O-methylglucose transport. 5. It was estimated that about 70% of the pH effect on the rates of glucose consumption may be due to the effect on sugar transport and the remainder to the effect on the activities of glycolytic enzymes.     

Cell surface changes and Rous sarcoma virus gene expression in synchronized cells

We have investigated whether cell surface changes associated with growth control and malignant transformation are linked to the cell cycle. Chicken embryo cells synchronized by double thymidine block were examined for cell-cycle-dependent alterations in membrane function (measured by transport of 2-deoxyglucose, uridine, thymidine, and mannitol), in cell surface morphology (examined by scanning electron microscopy), and in the ability of tumor virus gene expression to induce a transformation-specific change in membrane function. We reach the following conclusions: (a) The high rate of 2-deoxyglucose transport seen in transformed cells and the low rates of 2-deoxyglucose and uridine transport characteristic of density-inhibited cells do not occur in normal growing cells as they traverse the cell cycle. (b) Although there are cell cycle-dependent changes in surface morphology, they are not reflected in corresponding changes in membrane function. (c) Tumor virus gene expression can alter cell membrane function at any stage in the cell cycle and without progression through the cell cycle.     

Acute inhibition of insulin-stimulated glucose transport by the phosphatase inhibitor, okadaic acid.

Insulin is thought to exert its effects on cellular function through the phosphorylation or dephosphorylation of specific regulatory substrates. We have analyzed the effects of okadaic acid, a potent inhibitor of type 1 and 2A protein phosphatases, on the ability of insulin to stimulate glucose transport in rat adipocytes. Insulin and okadaic acid caused a 20-25- and a 3-6-fold increase, respectively, in the rate of 2-deoxyglucose accumulation by adipose cells. When added to cells previously treated with okadaic acid, insulin failed to stimulate 2-deoxyglucose accumulation beyond the levels observed with okadaic acid alone. Treatment of cells with okadaic acid did not inhibit the effect of insulin to stimulate tyrosine autophosphorylation of its receptor. These results indicate that okadaic acid potently inhibits the effects of insulin to stimulate glucose uptake and/or utilization at a step after receptor activation. To clarify the mechanism of inhibition by okadaic acid, the intrinsic activity of the plasma membrane glucose transporters was analyzed by measuring the rate of uptake of 3-O-methylglucose by adipose cells, and the concentration of adipocyte/skeletal muscle isoform of the glucose transporter (GLUT-4) in plasma membranes isolated from these cells. Insulin caused a 15-20-fold stimulation of 3-O-methylglucose uptake and a 2-3-fold increase in the levels of GLUT-4 detected by immunoblotting of isolated plasma membranes; okadaic acid caused a 2-fold increase in 3-O-methylglucose uptake, and a 1.5-fold increase in plasma membrane GLUT-4. Pretreatment of cells with okadaic acid blocked the effect of insulin to stimulate 3-O-methylglucose uptake and to increase the plasma membrane concentration of GLUT-4 beyond the levels observed with okadaic acid alone. These results indicate that the effect of okadaic acid to inhibit the effect of insulin on glucose uptake is exerted at a step prior to the recruitment of glucose transporters to the cell surface, and suggest that a phosphatase activity may be critical for this process.     

Regulation of the proliferative response in Rous sarcoma virus transformed chicken embryo fibroblasts by serum and multiplication-stimulating activity (MSA).

A temperature sensitive mutant of Rous sarcoma virus (tsNY68) was used to obtain cultures of quiescent virus-infected chicken embryo fibroblasts arrested by serum starvation at the non-permissive temperature. Upon shift to the permissive temperature, these cells enter the replicative cell cycle as evidenced by increases in 2-deoxyglucose uptake, 3H-thymidine incorporation and percent labeled nuclei. These changes occur in the absence of serum and the cells become morphologically transformed within eight to ten hours after the temperature shift. Entry into the S phase temporally resembles that of normal quiescent fibroblasts stimulated with serum. This experimental system was used to examine the proliferative response of transformed cells to serum and purified multiplication-stimulating activity (MSA) during the transition from the resting to the growing state. Data are presented which show that the presence of serum in the medium enhances the proliferative response of quiescent infected cells shifted to the permissive temperature over those shifted in the absence of serum. In contrast, the presence of MSA has no additional effect on the response exhibited by infected cells shifted to the permissive temperature in serum-free medium. Labeled MSA binding experiments show that this lack of response is not due to a loss of MSA receptors on the cell surface since transformed cells are still capable of binding MSA at the same level as normal cells. The results are consistent with the hypothesis that the set of biochemical events initiated by MSA in normal cells are turned on in infected cells shifted to the permissive temperature by the activation of the src gene product.     

Glucose transport in Entamoeba histolytica

That the uptake of glucose by the parasitic amoeba Entamoeba histolytica occurs by an equilibrative transport system is supported by the following observations. 1. The rate of glucose uptake is several orders of magnitude greater than the uptake by pinocytosis. 2. The uptake of glucose exhibits saturation kinetics, with K(m)=1.6mm and V(max.) ranging from 2 to 5mumol/min per ml of cells at 37 degrees C. 3. The glucose analogues 2-deoxyglucose, 3-O-methylglucose and d-xylose are transported by the glucose system although with much less affinity. Competitive inhibition was observed between pairs of substrates, with K(i) values for any sugar closely coincident with the corresponding K(m). 4. d-Xylose, a sugar not metabolized by the cells, equilibrated with 80% of the amoebal cell water. 5. Cells equilibrated with xylose exhibited countertransport of this sugar against its concentration gradient when another substrate was added to the medium. 6. Blocking of glycolysis by iodoacetate or F(-) has no immediate effect on transport. The presence of a glucose-transport system in E. histolytica contrasts with the situation found in the non-parasitic amoeba, where pinocytosis seems to be the only mechanism of solute uptake.     

SV40 transformation of mouse brain cells: critical role of gene A in maintenance of the transformed phenotype.

Brain cells derived from the NIH Swiss mouse strain have been established in tissue culture. Astrocytic neuroglial cells, identified by morphology and staining properties, predominate. The brain cell culture was successfully transformed with SV40 wild type virus and with a representative early (A239) and late (C219) mutant. When subjected to growth analysis the A239 transformant displayed selective loss of six characteristics of the transformed phenotype at the restrictive temperature (40.5 degrees C): doubling time, saturation density, ability to grow in low serum, efficiency of growth on plastic and on normal brain cell layers, and cloning in soft agar. Temperature shift experiment demonstrated the reversibility of the differences in saturation density. T-antigen was expressed at both temperatures. Alteration in uptake of 2-deoxyglucose was not a characteristic of the transformed phenotype. The cell lines may be utility in brain culture work and in studies on the mechanism of SV40 transformation.     

Quench-flow analysis reveals multiple phases of GluT1-mediated sugar transport

Standard models for carrier-mediated nonelectrolyte transport across cell membranes do not explain sugar uptake by human red blood cells. This means that either (1) the models for sugar transport are incorrect or (2) measurements of sugar transport are flawed. Most measurements of red cell sugar transport have been made over intervals of 10 s or greater, a range which may be too long to measure transport accurately. In the present study, we examine the time course of sugar uptake over intervals as short as 5 ms to periods as long as 8 h. Using conditions where transport by a uniform population of cells is expected to be monophasic (use of subsaturating concentrations of a nonmetabolizable but transported sugar, 3-O-methylglucose), our studies demonstrate that red cell sugar uptake is comprised of three sequential, protein-mediated events (rapid, fast, and slow). The rapid phase is more strongly temperature-dependent than the fast and slow phases. All three phases are inhibited by extracellular (maltose or phloretin) or intracellular (cytochalasin B) sugar-transport inhibitors. The rate constant for the rapid phase of uptake is independent of the 3-O-methylglucose concentration. The magnitude (moles of sugar associated with cells) of the rapid phase increases in a saturable manner with [3-O-methylglucose] and is similar to (1) the amount of sugar that is retained by red cell membrane proteins upon addition of cytochalasin B and phloretin and (2) the d-glucose inhibitable cytochalasin B binding capacity of red cell membranes. These results are consistent with the hypothesis that previous studies have both under- and overestimated the rate of erythrocyte sugar transport. These data support a transport mechanism in which newly bound sugars are transiently sequestered within the translocation pathway where they become inaccessible to extra- and intracellular water.     

Hexose sugar transport activity in a mouse fibroblast cell line temperature sensitive for expression of the transformed phenotype

A temperature-sensitive mouse fibroblast cell line was used to examine the relationship between hexose sugar uptake rates and the control of cell growth. The cell line used (ts-H6-15) is a derivative of SV-3T3 cells, exhibiting a transformed phenotype at 32°C and a normal phenotype at 39°C. For cells actively growing at either temperature, a marked decrease in the rate of 3-0-methyl-D-glucose (3-0-MeG) transport is observed as cell population density increases. At all cell population densities tested, 3-0-MeG transport rates (at a common assay temperature) were greater in H6-15 cells grown at 32°C than at 39°C, with the enhancement being maximal at the lowest cell densities. The effect of low serum-arrest on H6-15 cells revealed that cells growing at 39°C arrest in G1, while cells at 32°C stop more randomly throughout their cycle. Under conditions of low serum-arrest the rate of 3-0-MeG transport remained as high as in actively growing cells at both 32°C and 39°C. However, 2-deoxyglucose uptake rates were growth state-dependent at 39°C, indicating perhaps metabolic as well as membrane-level control of sugar accumulation. These results further demonstrate that rates of hexose sugar transport by themselves are not always absolutely correlated with rates of cell proliferation and, thus, may not be reliable predictors of cell growth potential.     

Transport of potassium, amino acids, and glucose in cells transformed by Rous sarcoma virus

Transport rates of a number of nutrients and ions have been surveyed in chicken embryo fibroblasts that were density inhibited, growing exponentially, or transformed by Rous sarcoma virus. All the transport systems examined displayed changes associated with changes in growth rate. The rate of ouabain-sensitive potassium transport declined in density-inhibited cells, and increased rapidly in response to serum stimulation. This transport system was regulated both by changes in the activity of the transporters and by the number of transporters in the cell membrane. The rate of transport of the amino acid analog alpha-aminoisobutyric acid declined when cells became density inhibited, but also showed alterations in regulation that were associated with malignant transformation. The rate of glucose transport displayed both growth state-related and transformation-specific changes. The increased rate of glucose transport seen in transformed cells is due to an increase in the number of glucose transporters in the cell membrane. Increased glucose transport is necessary for subsequent changes in glycolysis, and temporally precedes some of the changes in activity of glycolytic enzymes.