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.     

Insulin-dependent phosphorylation of the insulin receptor-protein kinase and activation of glucose transport in 3T3-L1 adipocytes

Insulin stimulates hexose transport and phosphorylation of the insulin receptor in monolayer cultures of intact 3T3-L1 adipocytes. To assess the phosphorylation state of the receptor in situ, cells were equilibrated with [32P]orthophosphate and then disrupted under denaturing conditions which preserved the phosphorylation state of the receptor established in the cell. The insulin receptor, isolated by lectin adsorption and two-dimensional nonreducing/reducing polyacrylamide gel electrophoresis, occurred as a single oligomeric species with an apparent alpha 2 beta 2 subunit composition. This oligomeric structure was not altered by treating cells with insulin. Only the beta-subunit of the receptor was phosphorylated; [32P]phosphoserine and [32P] phosphotyrosine were both identified in the beta-subunit from cells in the unstimulated state, but only [32P] phosphotyrosine increased in cells stimulated with insulin. Neither insulin-like growth factors I nor II stimulated insulin receptor beta-subunit phosphorylation, although both activated hexose transport. Upon the addition of insulin, [32P]orthophosphate incorporated into the beta-subunit increased 4.5-fold (7-fold with respect to [32P]tyrosine) and was complete within 1 min (t1/2 = 8 s). Following the removal of insulin from the monolayers, [32P]beta-subunit fell to the basal level (t1/2 = 2.5 min); there was no lag phase before either transition. The tyrosine protein kinase activity, measured in vitro with a model substrate, was higher with immunoaffinity-purified insulin receptor from insulin-stimulated cells than from cells in the basal state. Hexose transport rate, measured using 3-O-[methyl-14C]glucose, was half-maximally stimulated at 2 nM insulin. A 1-min latency period followed insulin addition, after which a 7-fold increase in the steady-state rate of hexose uptake was achieved within 5 min. Upon the removal of insulin, hexose transport continued at the stimulated steady-state rate for 2.5 min and then declined to the basal rate with a half-time of 8 min. These kinetic experiments in situ and protein kinase activity measurements in vitro support the hypothesis that beta-subunit phosphorylation is an intermediate step linking insulin binding to the increased glucose transport rate.     

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.     

Cell configuration-related control of vimentin biosynthesis and phosphorylation in cultured mammalian cells

The cell configuration-related control of a cytoskeletal protein (vimentin) expression was examined by varying cell shape between flat and spherical. Cultivation of cells in monolayer or in a spherical configuration on poly-2-hydroxyethylmethacrylate-coated plates revealed a preferential down regulation of vimentin synthesis during suspension culture. The mechanism(s) regulating the decrease in the expression of vimentin in spherical cells appears to be at the level of translation, because mRNAs extracted from monolayer and suspension-cultured cells were equally active in directing vimentin synthesis in the rabbit reticulocyte cell-free system. When after prolonged suspension culture, the cells were allowed to reattach and spread, vimentin synthesis recovered rapidly to the control monolayer rate. The phosphorylation of vimentin was also reduced dramatically during suspension culture. However, unlike the rapid recovery of vimentin biosynthesis upon reattachment (less than 6 h), the recovery in the rate of vimentin phosphorylation was much slower (greater than 20 h) and paralleled the recovery to the monolayer growth rate. Although the control of vimentin biosynthesis in suspension culture is a cell configuration-related process, the decrease in the rate of vimentin phosphorylation in suspension culture appears to be the result of the slower growth rate and may reflect the reported correlation between the rate of vimentin phosphorylation and the accumulation of cells in mitosis.     

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.     

The effects of sulfhydryl modifying reagents on nonhormonal and hormonally regulated hexose transport in cultured human skin fibroblasts

The effects of various sulfhydryl modifying reagents on hexose transport in cultured human skin fibroblasts were studied. H2O2 was observed to have no effect on 2-deoxy-D-glucose transport in serum-starved glucose-fed cells. The elevation of hexose transport rates in cells by glucose deprivation, insulin, or serum stimulation rendered them sensitive to H2O2. Hexose transport in glucose-deprived cells was inhibited 51-55% by 1-2 mM H2O2, while hexose transport in insulin or serum-stimulated glucose-fed cells was inhibited 45% and 46%, respectively. H2O2 inhibition was blocked or reversed by 8 mM dithiothreitol. N-ethyl-maleimide (NEM), a permeant, sulfhydryl reagent, elicited effects on hexose transport similar to those effected by H2O2 (i.e., in glucose-deprived and insulin-stimulated cells, inhibition of hexose transport was 44% and 23%, respectively). Impermeant sulfhydryl reagents such as dithio(bis)nitrobenzoic acid (DTNB) and N-iodoacetyl-N'-(5-sulfo-1-naphthly-ethylenediame (1,5,-I-AEDANS) had no inhibitory effect on hexose transport under any conditions (i.e., glucose-fed, glucose-deprived, and insulin-stimulated cells). DTNB and 1,5-I-AEDANS afforded no protection from the action of H2O2 on hexose transport. The data suggest that the sensitive sites are thiol in nature and are located at an intramembrane or intracellular site and probably not exofacial.     

Selective high metabolic lability of uridine triphosphate in response to glucosamine feeding of untransformed and polyoma virus-transformed hamster fibroblasts

Derepression of hexose transport in a line of Syrian hamster fibroblasts (Nil) and polyoma-transformed (PyNil) hamster fibroblasts is obtained when cells are either starved for glucose or fed with fructose as the only hexose source. D-glucosamine feeding of these cells does not alter the repressed state with regard to hexose transport. High, derepressed rates of galactose transport were changed to low, repressed rates, within 18 hours of refeeding glucose-starved cells with D-glucosamine as the only hexose source. Nil and PyNil cells, when cultured in the presence of D-glucosamine, undergo rapid reductions in total cellular uridine 5′-triphosphate (UTP) pool sizes. By contrast, the total cellular pools of adenosine 5′-triphosphate, guanosine 5′-triphosphate, and cytosine 5′-triphosphate (ATP, GTP, and CTP) were only moderately affected by the treatment of the cells with glucosamine. The metabolic drain of the UTP pools in PyNil cells was much more pronounced than in the untransformed cells. The larger and more rapid metabolic lability of UTP pools in the transformed cells may be the primary reason for the selective toxicity of glucosamine on tumor cells. A comparison of the effects of glucosamine on hexose-starved Nil and PyNil cells demonstrated that only the untransformed cells were able to utilize glucosamine to increase the hexose starvation-depleted pools of all nucleoside triphosphates. Accumulation of UDP-glucosamine and UDP-N-acetylglucosamine followed the reduction in the UTP pools. Inhibition of protein synthesis by cycloheximide during glucosamine feeding led to higher levels of UDP-glucosamine and UDP-N-acetylglucosamine accumulation. It is suggested that the drain of UTP pools during glucosamine treatment proceeds through the formation of the UDP-aminosugars which turn over due to the action of intracellular UDP-aminosugar pyrophosphatase activities.     

Transport changes associated with growth control and malignant transformation.

We can distinguish two classes of membrane transport changes in cultured cells: (a) growth-rate contingent changes are those which occur in coordination with the onset of density-dependent inhibition of growth; (b) transformation-specific changes are those which occur when cells become transformed, and which can be detected even when normal and transformed cells are growing at the same rate. Growth-rate contingent changes include the density-dependent changes in phosphate, nucleoside, glucose, amino acid, and potassium transport. Only one transformation-specific transport change has been found in Rous-transformed chicken embryo fibroblasts: an increased rate of hexose transport. The variation in potassium transport are associated with variations in the number of ouabain binding sites in the membrane. The molecular basis for changes in the rate of hexose transport is unknown, although gross changes in membrane bilayer composition and "fluidity" seem not to be involved. In analyzing the regulation of hexose transport activity, we find that decreased cAMP may play a role in the transformation-specific increase in hexose transport, but that fibrinolytic activity is not necessary.     

Kinetic characteristics and regulation of hexose transport in a galactokinase-negative Chinese hamster fibroblast cell line: a good model for studies on sugar transport in cultured mammalian cells

We report the kinetic characteristics for D-galactose, 2-deoxy-D-glucose and 3-O-methyl-D-glucose transport in a galactokinase null-allele mutant of a Chinese hamster V79 cell line. GalKl cells exhibited a Km and Vmax for D-galactose, 2-deoxy-D-glucose, and 3-O-methyl-D-glucose transport of 8.6 +/- 2.6 mM and 26.1 +/- 7.2 nmol/mg p/min, 4.1 +/- 1.2 mM and 40.3 +/- 9.5 nmol/mg p/min, and 7.01 +/- .85 mM and 11.6 +/- 4.8 nmol/mg p/30 s, respectively. Nonsaturable hexose uptake was determined using cytochalasin B inhibition of galactose uptake (89.6 +/- 3.7% of galactose uptake was cytochalasin B inhibitable) and L-glucose uptake (7.5% of the galactose uptake). D-Galactose was not metabolized and effluxed rapidly from preloaded cells. The Kls for the inhibition of D-galactose transport were 4.5 +/- 2.5 mM for D-glucose, 7.0 +/- 2.0 mM for 2-deoxy-D-glucose, 6 mM for 2-deoxy-D-galactose and 6.0 +/- 0.6 mM for 3-O-methyl-D-glucose. This indicates the operation of a single common carrier. The hexose transport rate decreased 50-60% after 24 h serum deprivation. Addition of insulin was shown to increase hexose transport (more than twofold) in serum-deprived cells. Hexose transport rates increased substantially in glucose-deprived, D-fructose- or D-galactose-fed cells as compared to glucose-fed cells. Since GalKl does not metabolize galactose, the hexose transport increases induced by feeding cells galactose suggest that carrier interaction with ligand is not a significant factor in transport regulation in GalKl. The kinetic and regulatory characteristics of D-galactose transport in the GalKl cell line indicate that this system is a good model to study sugar transport from a mechanistic and regulatory point of view.     

Morphological transformation, autonomous proliferation and colony formation by chicken heart mesenchymal cells infected with avian sarcoma, erythroblastosis and myelocytomatosis viruses

Normal chicken heart mesenchymal cells at low density in monolayer culture in plasma-containing medium have a polygonal shape and are proliferatively quiescent. The combination of epidermal growth factor and insulin at hyperphysiological concentration, an insulin-like growth factor surrogate, causes these cells to assume a fusiform shape and to increase 40-fold in number during four days of incubation. These mitogenic hormones do not, however, induce normal chicken heart mesenchymal cells to form colonies in agarose suspension culture. Chicken heart mesenchymal cells infected with the Schmidt-Ruppin or Prague-A strains of Rous sarcoma virus or with the Fujinami or Y73 avian sarcoma viruses assume spindle and round shapes, increase 50-100 fold in number during four days of monolayer culture in the absence of mitogenic hormones and form macroscopic colonies during 3-4 days of agarose suspension culture. The autonomous (mitogenic hormone-independent) proliferation, in monolayer culture, of cells infected with temperature-sensitive transformation mutants of Rous sarcoma virus (tsNY68, tsNY72, tsLA24, tsLA29) is temperature-sensitive. Chicken heart mesenchymal cells infected with avian erythroblastosis virus assume spindle shapes and proliferate in monolayer culture at a rate comparable to that of sarcoma virus-infected cells but do not, however, form colonies in agarose suspension culture. Cells infected with the myelocytomatosis virus MC29 assume stellate shapes and increase 18-fold in number during four days of monolayer culture. Cells infected with the myelocytomatosis virus MH2 assume fusiform shapes and increase fourfold in number during four days of monolayer culture. Neither MC29 nor MH2 renders chicken heart mesenchymal cells capable of colony formation in agarose suspension culture. Infection with avian leukosis viruses (RAV-1, RAV-2, RPL-42) or with transformation-defective mutants of Rous sarcoma virus (tdNY105, 107, 109) does not affect the morphology or proliferative behavior of chicken heart mesenchymal cells. Monolayer culture of chicken heart mesenchymal cells in plasma-containing medium appears, therefore, to define the ability of onc genes of acute transforming avian retroviruses to induce autonomous (mitogenic hormone-independent) cell proliferation, the essential characteristic of neoplasia. The differences in transformed morphology and rates of autonomous proliferation between cells infected with different acute transforming retroviruses probably reflects differences in the modes of action of the transforming proteins encoded by the onc genes of the respective viruses.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hexose transport in human myoblasts.

The present investigation reports on the hexose transport properties of human myoblasts isolated from normal subjects and from patients with Duchenne muscular dystrophy (DMD). Similar to rat myoblast L6, normal human myoblasts possess a high- (HAHT) and a low- (LAHT) affinity hexose transport system. The non-metabolizable hexose analogue, 2-deoxyglucose, is preferentially taken up by HAHT. The transport of this analogue is the rate-limiting step in the uptake process. This human myoblast HAHT is also similar to that of the rat myoblast in its substrate specificity and in response to the energy uncouplers, cytochalasin B and phloretin. The human myoblast LAHT resembles that of rat myoblast in its insensitivity to energy uncouplers, and in its transport affinity and capacity for 3-O-methyl-D-glucose. Although DMD myoblasts resemble their normal counterpart in their ability to differentiate, they differ significantly in their hexose transport properties. In addition to HAHT and LAHT present in normal human myoblast, DMD myoblasts contain a super-high-affinity hexose transport system (SHAHT). SHAHT can be detected only at very low substrate concentrations. It differs from HAHT not only in its much higher transport affinity, but also in its response to the traditional hexose transport inhibitors. For example, SHAHT can be activated by cytochalasin B and phlorizin, whereas it is more sensitive to inhibition by phloretin. Unlike HAHT, energy uncouplers are found to be ineffective in inhibiting SHAHT. It should be mentioned that SHAHT cannot be detected in myoblasts isolated from patients with other types of myopathy. The present study serves to demonstrate that more than one hexose transport system is operating in human skeletal muscle cells, as found in other cell types.     

Abnormal phenotype of in vitro dermal fibroblasts from patients with Pseudoxanthoma elasticum (PXE)

Pseudoxanthoma elasticum (PXE) is a genetic connective tissue disease, whose gene and pathogenesis are still unknown. Dermal fibroblasts from patients affected by PXE have been compared in vitro with fibroblasts taken from sex and age-matched normal individuals. Cells were grown and investigated in monolayer, into three-dimensional collagen gels and in suspension. Compared with normal cells, PXE fibroblasts cultured in monolayer entered more rapidly within the S phase and exhibited an increased proliferation index; on the contrary, similarly to normal fibroblasts, PXE cells did not grow in suspension. Furthermore, compared with normal fibroblasts, PXE cells exhibited lower efficiency in retracting collagen type I lattices and lower adhesion properties to collagen type I and to plasma fibronectin. This behavior was associated with higher expression of integrin subunits alpha2, alpha5, alphav, whereas beta1 subunit as well as alpha2beta1 and alpha5beta1 integrin expression was lower than in controls. Compared to controls, PXE fibroblasts had higher CAM protein expression in accordance with their high tendency to form cellular aggregates, when kept in suspension. The demonstration that PXE fibroblasts have altered cell-cell and cell-matrix interactions, associated with modified proliferation capabilities, is consistent with the hypothesis that the gene responsible for PXE might have a broad regulatory role on the cellular machinery.     

Density-dependent changes in hexose transport, glycolytic enzyme levels, and glycolytic rates, in uninfected and murine sarcoma virus-transformed rat kidney cells

In normal rat kidney (NRK) cell cultures, increased cell density results in a decrease in the rates of hexose transport, glucose utilization, and lactate production and an increase in the level of hexokinase activity. A murine sarcoma virus (Kirsten)-transformed cell line (KNRK) showed little or no density-dependent variation in sugar uptake, glucose consumption, or lactate production. On the other hand, hexokinase, phosphofructokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase activities were elevated in dense transformed cultures as compared to sparse or uninfected cultures. In another virus-transformed cell line (ts339/NRK) exhibiting temperature-dependent morphology, growth pattern, and transport of 2-deoxy- -glucose, the levels of glycolytic enzyme activity were related to cell density but not to the culture temperature. The lack of correlation between glycolytic enzyme activity and lactate production by either uninfected or murine sarcoma virus-transformed cultures supports the suggestion that enhanced growth and/or hexose transport capacity rather than elevated glycolytic enzyme activity are responsible for the increased rate of lactate production by virus-transformed NRK cells.     

Isolation and characterization of hexose transport mutants in L6 rat myoblasts

A method for the selection and isolation of hexose transport mutants in undifferentiated rat myoblast L6 cells is reported; 2-deoxy-D-glucose (2-DOG)-and 2-deoxy-2-fluoro-D-glucose (2FG)-resistant mutants were selected after mutagenization of L6 cells with ethyl methanesulfonate. Of these, D18 and D23 (selected with 0.1 mM 2-DOG) and F72 and F76 (selected with 0.1 mM 2FG) exhibited the lowest hexose transport activity. Uptake of 0.06 mM 2-DOG, 2FG, or 3-O-methyl-D-glucose (3-OMG) by mutants grown in fructose medium supplemented with 0.05 mM 2FG was about four- to five-fold lower than the parental L6 cells. These mutants contain normal levels of ATP and glycolytic enzyme activities. They also exhibit normal transport activities for alpha-aminoisobutyric acid and fructose. Furthermore, hexose transport was observed to be decreased in plasma membrane vesicles prepared from these mutants. Kinetic analysis of 2-DOG and 3-OMG transport in mutant F72 demonstrated that the Vmax for 2-DOG uptake was significantly reduced, whereas the Vmax for 3-OMG transport was not affected. In all cases, the affinity for these hexose analogues was unaffected. In addition mutant F72 was found to be only slightly affected by treatment with various energy inhibitors and sulfhydryl reagents. The results suggest that this mutant is defective in, or has low levels of, a plasma membrane component(s) involved in the high-affinity hexose transport system.     

Hexose transport regulation in cultured hamster cells

Hamster (nil) cells maintained overnight in culture medium containing cycloheximide and either glucose or fructose exhibit strikingly different rates of hexose transport and metabolism (i.e., uptake). Pretreatment of cultures with sulfhydryl reagents makes it possible to determine initial transport rates for a physiological sugar such as galactose which is a catabolite in hamster cells. Using galactose transport as a model, hexose uptake enhancements can now be shown to be due almost entirely to increase in the rate of the transport step. The transport regulation can best be accounted for by a model comprised of 2 antagonizing mechanisms. This model involves turnover of transport carriers as well as inhibitory units (“regulators”). The experimental as well as the theoretical model may also apply to the well-known uptake enhancements observed in oncogenically 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.     

Endogenous regulation of 2-deoxyglucose uptake in C6 glioma cells correlates with cytoskeleton-mediated changes of surface morphology

The cellular basis of the membrane-limited state of glucose utilization and the mechanism of the endogenous regulation of hexose uptake in dense monolayers of C6 glioma cells were investigated. In an earlier study, it was shown that at high rates of glucose transport and phosphorylation combined with the inhibition of glycolytic adenosine triphosphate (ATP) production by iodoacetate, an endogenous regulatory response occurred that resulted in rapid, periodic variations of the glucose uptake rates (Lange et al., 1982). Similar time-dependent periodic changes of uptake rates also occurred during incubation of C6 glioma cells with 2 mM 2-deoxyglucose (2-DG) without pretreatment of the cells with iodoacetate. These changes were accompanied by variations of the intracellular ATP content, by distinct alterations of the shape and arrangement of microvilli and lamellae (lamellipodia) on the cell surface, and by changes of the cytoskeletal F-actin content. Because the changes of 2-DG uptake rates occurred independent of the intracellular 2-DG concentration, the bulk of this 2-DG pool was assumed to be localized apart from the membranal transport sites. Downregulation of 2-DG uptake appeared to be triggered by a rapid decrease of a small pool of the cellular ATP involved in the phosphorylation of transported hexose. Scanning and transmission electron microscopic observations of cells fixed in different states of the endogenous uptake regulation supported the assumption that the interior of lamellae and microvilli may represent a small entrance compartment for transported hexoses in which occurred the observed close coupling between hexose transport and phosphorylation as well as the rapid variations of ATP content. Hexose uptake is supposed to be regulated by cytoskeleton-mediated changes of volume and diffusional accessibility of this compartment, modulating the degree of its metabolic coupling with the cytoplasmic main compartment.     

Inhibition by forskolin of insulin-stimulated glucose transport in L6 muscle cells.

The cardioactive diterpene forskolin is a known activator of adenylate cyclase, but recently a specific interaction of this compound with the glucose transporter has been identified that results in the inhibition of glucose transport in several human and rat cell types. We have compared the sensitivity of basal and insulin-stimulated hexose transport to inhibition by forskolin in skeletal muscle cells of the L6 line. Forskolin completely inhibited both basal and insulin-stimulated hexose transport when present during the transport assay. The inhibition of basal transport was completely reversible upon removal of the diterpene. In contrast, insulin-stimulated hexose transport did not recover, and basal transport levels were attained instead. This effect of inhibiting (or reversing) the insulin-stimulated fraction of transport is a novel effect of the diterpene. Forskolin treatment also inhibited the stimulated fraction of transport when the stimulus was by 4 beta-phorbol 12,13-dibutyrate, reversing back to basal levels. Half-maximal inhibition of the above-basal insulin-stimulated transport was achieved with 35-50 microM-forskolin, and maximal inhibition with 100 microM. Forskolin did not inhibit 125I-insulin binding under conditions where it caused significant inhibition of insulin-stimulated hexose transport. Forskolin significantly elevated the cyclic AMP levels in the cells; however its inhibitory effect on the above basal, insulin-stimulated fraction of hexose transport was not mediated by cyclic AMP since: (i) 8-bromo cyclic AMP and cholera toxin did not mimic this effect of the diterpene, (ii) significant decreases in cyclic AMP levels caused by 2',3'-dideoxyadenosine in the presence of forskolin did not prevent inhibition of insulin-stimulated hexose transport, (iii) isobutylmethylxanthine did not potentiate forskolin effects on glucose transport but did potentiate the elevation in cyclic AMP, and (iv) 1,9-dideoxyforskolin, which does not activate adenylate cyclase, inhibited hexose transport analogously to forskolin. We conclude that forskolin can selectively inhibit the insulin- and phorbol ester-stimulated fraction of hexose transport under conditions where basal transport is unimpaired. The results are compatible with the suggestions that glucose transporters operating in the stimulated state (insulin or phorbol ester-stimulated) differ in their sensitivity to forskolin from transporters operating in the basal state, or, alternatively, that a forskolin-sensitive signal maintains the stimulated transport rate.     

Hexose transport in normal and SV40-transformed human endothelial cells in culture

The mechanism of glucose entry into human vascular endothelial cells was studied in monolayer cultures of normal (primary) and virally (SV40) transformed umbilical vein endothelium. Radioisotopic uptake studies with the glucose analogues 2-deoxy-D-glucose, and 3-O-methyl-D-glucose, and the nonmetabolizable stereoisomer L-glucose, indicated the presence of a saturable, stereospecific hexose carrier mechanism in both cell types. In other experiments with D-glucose and 3-O-methyl-D-glucose, the phenomenon of countertransport was demonstrable. Hexose transport was not affected by KCN, dinitrophenol, or ouabain, but was inhibited by phloretin and phlorizin in a pattern consistent with facilitated diffusion. Kinetic constants were obtained for both 2-deoxy-D-glucose and 3-O-methyl-D-glucose uptake. Similar Km values (range, 3.3-4.7 mM) were noted with normal and transformed cells, whereas the apparent Vmax was 0.56 nmol/microliter cytosol/minute for primary cells and 1.7-2.5 nmol/mu cytosol/minute for transformed cells. Under standard culture conditions, as well as following 18 hours of serum deprivation, insulin at concentrations up to 10(-5) M did not appear to influence hexose uptake in either cell type. Metabolism of 14C(U)-D-glucose to 14CO2 also was not stimulated by insulin. The presence of an insulin-insensitive, facilitated transport system for glucose in vascular endothelium has relevance for glucose metabolism in this tissue, and potentially for the association of certain vascular diseases (e.g., diabetic microangiopathy, atherosclerosis) with altered glucose homeostasis.     

Increased insulin-insensitive glucose transport in polymorphonuclear leukocytes from non-insulin-dependent diabetic patients.

We studied the transport rate of a non-metabolizable hexose analogue, 3-O-methyl-D-glucose, in polymorphonuclear leukocytes (insulin-insensitive cells) from patients with untreated non-insulin-dependent diabetes mellitus. The mean glucose transport rate was significantly elevated in the diabetic patients compared with healthy controls (13.3 +/- 3.7 vs 10.4 +/- 2.5 fl/cell.sec, mean +/- SD, p less than 0.01). In the diabetic subjects, glucose transport rates were positively correlated with HbA1c levels (r = 0.563, p less than 0.01) but had no relations with ambient plasma glucose concentrations. Short-term incubation with 20 mM D-glucose had no effect on glucose transport in those cells. When glucose transport rates, HbA1c and fasting plasma glucose levels were simultaneously measured at weekly intervals over a four-week period in three diabetic subjects, the alterations in transport rates generally paralleled the changes observed in HbA1c levels rather than plasma glucose concentrations. It can be concluded that unlike insulin-sensitive cells such as adipocytes and muscle, glucose transport in human polymorphonuclear leukocytes, which are insulin insensitive cells, is increased in patients with non-insulin-dependent diabetes mellitus. Long-term, not short-term, derangement of glucose metabolism seems to be associated with increased glucose transport rate found in those patients.