Current status of the thiol redox model for the regulation of hexose transport by insulin.

Data obtained over the last two years pertinent to the thiol redox model for the modulation of hexose transport activity by insulin is summarized. The model proposes that activation of hexose transport in fat cells involves sulfhydryl oxidation to the disulfide form in a key protein component of the fat cell surface membrane. Theoretically, the rapid activation of transport by insulin may involve either the conversion of inactive membrane carriers to the active form as originally proposed, or the conversion of a low Vmax transport system to a high Vmax form. The present experiments showed that the percent inhibition of insulin-activated transport rates by submaximal levels of cytochalasin B was decreased compared to its effects on basal transport. Treatment of fat cells with N-ethylmaleimide inhibited cytochalasin B action but not transport activity. When insulin or the oxidant vitamin K5 was added to cells 5 minutes before the N-ethylmaleimide, the elevated transport activity was also resistant to the sulfhydryl reagent, but cytochalasin B retained its potent inhibitory effect on transport. The data demonstrate that unique properties characterize basal versus insulin-activated transport activity with respect to the sensitivity of cytochalasin B action to sulfhydryl blockade in isolated fat cells. The data are consistent with the concept that activation of transport activity reflects the conversion of a reduced (sulfhydryl) system characterized by a low Vmax to an oxidized (disulfide), high Vmax transport system.     

Restoration of normal morphology, adhesion and cytoskeleton in transformed cells by addition of a transformation-sensitive surface protein.

Transformed cells lack a large, external, transformation-sensitive (LETS) glycoprotein which is a major surface component of their normal counterparts. Addition of LETS glycoprotein isolated from normal cells to transfomed cells restores certain morphological features and adhesive properties characteristic of normal cells. LETS protein is detected on the cell surface both by iodination using lactoperoxidase and by immunofluorescent staining. The surface distribution pattern detected by immunofluorescence is strikingly similar to that of normal cells. After addition of LETS protein, transformed cells also exhibit well defined actin cables which are not seen in untreated, transformed cells. All these alterations can be blocked by treating LETS protein with specific antisera or by subjecting it to mild trypsinization prior to addition to transformed cells. The effects are rapidly reversible by mild trypsinization, which removes the added LETS protein. The high rate of uptake of 2-deoxyglucose, characteristic of transformed cells, is not affected by LETS protein. These results suggest that LETS protein may have a role in cell attachment and spreading, and affect the organization of cytoskeleton.     

Surface distribution of LETS protein in relation to the cytoskeleton of normal and transformed cells

The organization of LETS protein on the surface of NIL8 hamster cells has been examined by immunofluorescence staining. The distribution of LETS protein was found to depend on the culture conditions; in subconfluent, low-serum arrested cultures the LETS protein is predominantly located at the cell-substrate interface and also in regions of cell-cell contact, whereas in dense cultures the cells are surrounded by a network of LETS protein fibrils. Transformed derivatives of these cells exhibit only sporadic staining for LETS protein, in the form of short intercellular bridges. Agents that cause alterations in cell shape and cytoplasmic filaments have been used to explore the relationship of LETS protein to the internal cytoskeletal elements. Reciprocally, perturbations of the cell surface were examined for their effects on internal filaments. The arrangement of microtubules seems to be unrelated to the presence of LETS protein in the cells studied. Actin microfilament bundles and LETS protein respond in a coordinate fashion to some perturbants but independently with respect to others. The patterns of staining for LETS protein are consistent with an involvement in cell-to-cell and cell-to-substrate adhesion.     

Cell surface and metabolic labelling of the proteins of normal and transformed chicken cells.

We have studied the surface proteins of normal and transformed chick cells using four-labelling techniques with different specificities, (a) lactoperoxidase catalysed iodination (b) galactose oxidase/B3H4 (c) pyridoxal phosphate/B3H4 and (d) periodate/B3H4. All methods labelled a large external transformation-sensitive (LETS) protein, in agreement with previous studies. In addition, using galactose oxidase and periodate labelling techniques, we present evidence which suggests that the transformed cell surface glycoproteins are more sialylated. The LETS protein was also labelled with (14C) glucosamine and after trypsinization a small band of identical molecular weight to LETS remained, possibly representing an internal pool of the protein. In contrast LETS protein labelled with (3H) fucose was completely removed by trypsin, suggesting that the internal pool of the protein is incompletely glycosylated. Evidence is also presented to show that although the level of the protein is drastically reduced at the transformed cell surface, it is still synthesised and shed into the medium.     

Role of disulfide bonds in the attachment and function of large, external, transformation-sensitive glycoprotein at the cell surface

Reduction of disulfide linkages by dithiothreitol removes LETS (large, external, transformation-sensitive) protein from the cell surface. This process is dependent upon the concentration of dithiothreitol and the time and temperature of reaction. At 0 degrees C the release of LETS protein by dithiothreitol is completely blocked, but this is apparently not due to a requirement for metabolic energy. At this temperature, reduction of LETS protein is incomplete. These results suggest that intact disulfide bonds are involved in the retention of this protein on the cell surface. Furthermore, reduction of purified LETS protein interferes with its ability to confer flattened morphology and increased adhesivity when added to transformed cells. It appears, therefore, that disulfide bonds are functionally important at the cell surface.     

Effects of LETS glycoprotein on cell motility

Addition of LETS glycoprotein to normal or transformed cells produces increased migration of the cells, as determined by formation of phagokinetic tracks on gold particle-coated coverslips. These tracks arise by a combination of phagocytosis of the gold particles and cellular migration. Increased motility is also evident on plastic in the absence of gold particles. The added LETS protein attaches to the cells in a fibrillar network, and binding is greater to normal than to transformed cells. The effects of LETS protein on migration are consistent with its effects on cell adhesion, morphology and cytoskeleton, and have potential implications for the determination of cellular migration in vivo.     

Characterisation of monoclonal antibodies which specifically recognise the human erythrocyte glucose transport protein

Two monoclonal antibodies (mabs) of subclass IgG1 have been raised against the human erythrocyte glucose transport protein. The mabs bound to the purified glucose transporter in both its membrane-bound and detergent-solubilised forms. However, they exhibited little or no binding to the detergent-solubilised nucleoside transport protein, which is present as a minor contaminant in the glucose transport protein preparation. Both mabs inhibited the binding of cytochalasin B to the glucose transport protein, reducing the affinity of this binding by greater than 2-fold. Each mab labelled the transporter polypeptide on Western blots both before and after treatment of the protein with endoglycosidase F, indicating that the epitopes recognised were located on the protein moiety of the glycoprotein. However, the mabs did not bind to the large fragments produced by tryptic or chymotryptic digestion of the native protein, although both mabs were shown to bind to sites on the cytoplasmic surface of the erythrocyte membrane.     

Lack of correlation between the decreased expression of cell surface LETS protein and tumorigenicity in human cell hybrids

An analysis of the correlation between tumorigenicity and the loss of expression of the large external transformation-sensitive glycoprotein (LETS) was performed on human cell hybrids and their respective normal and tumorigenic parental cell lines. The distribution of cell surface LETS protein in a series of cell lines was examined by both specific immunofluorescent staining and by gel electrophoresis of lactoperoxidase-catalyzed, iodinated cell surface proteins. The tumorigenicity of these cell lines was assayed in nude mice. Although the series of cell lines studied provided a broad spectrum of LETS protein expression, both quantitatively and qualitatively, there does not appear to be a correlation between tumorigenicity and decreased expression of the LETS protein.In a series of transformed, nontumorigenic hybrids, the LETS protein expression was found to be altered with respect to both decreased organizational complexity and decreased content. These hybrids continue to express a number of other transformed phenotypes. Conversely, a number of tumorigenic hybrids continue to express relatively high levels of LETS protein when compared with nontumorigenic hybrids. Thus an alteration in LETS protein expression by itself, or in concert with a spectrum of other transformation properties, does not appear to be a sufficient requirement for tumorigenicity and lends further support to an apparent separate control of the transformed versus tumorigenic phenotype.     

Reaction of an exofacial sulfhydryl group on the erythrocyte hexose carrier with an impermeant maleimide. Relevance to the mechanism of hexose transport

The presence of a reactive exofacial sulfhydryl on the human erythrocyte hexose carrier was used to test several predictions of the alternating conformation or one-site model of transport. The cell-impermeant glutathione-maleimide-I (GS-Mal) irreversibly inhibited hexose entry by decreasing the transport Vmax. This effect was potentiated by phloretin and maltose but decreased by cytochalasin B, indicating that under the one-site model the external sulfhydryl is on the outward-facing carrier but that it does not overlap with the exofacial substrate-binding site. Incubation of erythrocytes with maltose competitively inhibited the binding of [3H]cytochalasin B to the inward-facing carrier (Ki = 40 mM). Furthermore, both equilibrium cytochalasin B binding and its photolabeling of the band 4.5 carrier protein were decreased in ghosts prepared from GS-Mal-treated cells. Thus induction of an outward-facing carrier conformation with either maltose or GS-Mal caused the endofacial substrate-binding site to disappear. Dose-response studies of GS-Mal treatment of intact cells suggested that some functional carriers lack a reactive external sulfhydryl, which can be partially regenerated by pretreatment with excess cysteine. These data provide direct support for the one-site model of transport and further define the role of the external sulfhydryl in the transport mechanism.     

Effects of cytoskeleton-disrupting agents on tyrosine transport into cultured bovine adrenal chromaffin cells

The effects of various cytoskeleton-disrupting agents on tyrosine transport into chromaffin cells were examined to assess the possibility of cytoskeleton involvement in the regulation of precursor supply for catecholamine synthesis. Tyrosine transport was markedly increased by cytochalasin B. Vinblastine also stimulated tyrosine transport, although its effect was less pronounced than that of cytochalasin B. While colchicine failed to cause any significant increase in the transport under the same conditions. These results therefore suggest a possible role of microfilaments as a factor regulating tyrosine transport into chromaffin cells.     

Alteration in cell surface LETS protein during myogenesis.

Cell surface alterations during myogenesis have been investigated in Yaffe's myogenic cell line L8, using indirect immunofluorescence with an antibody against the large external transformation-sensitive (LETS) protein. The immunofluorescent technique reveals a susbstantial alteration in the distribution of this surface antigen. With the prefused myoblasts, LETS protein is dispersed all over the cell surface; following myoblast fusion, this pattern is markedly changed. All of the fibril-like surface LETS protein disappears, and in some myotubes, discrete clusters of LETS protein become conspicuous. By use of radioimmunological assay, the total LETS protein is quantitatively reduced upon myoblast fusion.     

Inhibition of hexose transport by adenosine derivatives in human erythrocytes

The human erythrocyte membrane carriers for hexoses and nucleosides have several structural features in common. In order to assess functional similarities, the effects of adenosine derivatives on hexose transport and cytochalasin B binding sites were studied. Adenosine inhibited zero-trans uptake of 3-O-methylglucose half-maximally at 5 mM, while more hydrophobic adenosine deaminase-resistant derivatives were ten- to 20-fold more potent transport inhibitors. However, degradation of adenosine accounted for very little of this difference in potency. Hexose transport was rapidly inhibited by N6-(L-2-phenylisopropyl)adenosine at 5 degrees C in a dose-dependent fashion (EC50 = 240 microM), to lower the transport Vmax without affecting the Km. A direct interaction with the carrier protein was further indicated by the finding that N6-(L-2-phenylisopropyl)adenosine competitively inhibited [3H]cytochalasin B binding to erythrocytes (Ki = 143 microM) and decreased [3H]cytochalasin B photolabeling of hexose carriers in erythrocyte ghosts. The cross-reactivity of adenosine and several of its derivatives with the hexose carrier suggests further homologies between the carriers for hexoses and nucleosides, possibly related to their ability to transport hydrophilic molecules through the lipid core of the plasma membrane.     

Effects of cocultivation with transformed cells on surface proteins of normal cells.

Virally transformed fibroblasts do not have on their surface a major protein (large external transformation-sensitive, LETS) which is present in normal cells. Cocultivation of the transformed cells with normal cells whose surface proteins have been prelabelled induces an accelerated release of the LETS protein from the normal cells. We have investigated various conditions which affect this phenomenon. Our results show that alteration of cell surface proteins by cocultivation with the transformed cells is time and dose-dependent and requires cell contact. Serum was depleted at least 99% of plasminogen by affinity chromatography and used in the cocultivation experiments. It was found that activation of plasminogen was not required for the accelerated turnover of the LETS protein. Other diffusible proteases are also unlikely to be involved. The possibility that transformed cells have a membrane bound activity is discussed. The role of plasminogen activation was also tested for its relevance in transformation related proteolysis, growth and morphology of cells.     

INHIBITION OF PHAGOCYTOSIS AND PLASMA MEMBRANE MOBILITY OF THE CULTIVATED MACROPHAGE BY CYTOCHALASIN B : Role of Subplasmalemmal Microfilaments

Functional and morphologic effects of cytochalasin B on the cultivated macrophage were examined to determine the basis for plasma membrane movements of the type required for endocytosis and/or spreading on a substratum. Inhibition of phagocytosis and changes in cell shape by cytochalasin B exhibited nearly identical dose-response curves requiring 2–5 x 10^-6 M and 1–2 x 10^-5 M cytochalasin B to inhibit these functions by 50% and 100%, respectively. In contrast, hexose transport was ten times more sensitive to the drug requiring 2–3 x 10^-7 M cytochalasin B to achieve 50% inhibition of 2-deoxyglucose uptake. Inhibition of phagocytosis and changes in cell shape could not be explained solely by drug effects on hexose transport. Analysis of serial thin sections showed that cytochalasin B doses inhibitory for hexose transport had no effect on distribution or organization of either of the two subplasmalemmal microfilament types. However, cytochalasin B concentrations (2.0 x 10^-5 M) that inhibited phagocytosis and altered cell shape disorganized and/or disrupted oriented bundles of 40–50-Å subplasmalemmal microfilaments, but had no effect on the microfilamentous network. Comparative dose-response studies showing positive correlations among cytochalasin B effects on phagocytosis, changes in cell shape, and alterations in oriented subplasmalemmal microfilament bundles provide additional support for the hypothesis that microfilamentous structures play a role in translocation of plasma membrane required for endocytosis and cell motility.     

Binding of [3H]ctyochalasin B and [3H]colchicine to isolated liver plasma membranes.

The binding to isolated hepatocyte plasma membranes of radioactively labelled inhibitors of microfilamentous and microtubular protein function ([3H]cytochalasin B and [3H]colchicine, respectively) was studied as one means of assessing the degree of association of these proteins with cell surface membranes. [3H]Cytochalasin B which behaved identically to the unlabelled compound with respect to binding to these membranes was prepared by reduction of cytochalasin A with NaB3H4. The binding was rapid, readily reversible, proportional to the amount of membrane and relatively insensitive to changes of pH or ionic strength. At 10(-6) M [3H]cytochalasin B, glucose of p-chloromercuribenzoate, an inhibitor of glucose transport inhibited binding by about 20%; treatment of membranes with 0.6 M KI which depolymerizes F actin to G actin caused about 60% inhibition of binding. These two types of inhibition were additive indicating two separate classes of binding sites, one associated with sugar transport and one with microfilaments. Filamentous structures with the diameter of microfilaments (50 A) were seen in electron micrographs of thin sections of the membranes. At concentrations greater than 10(-5) M [3H]cytochalasin B, binding was proportional to drug concentration, characteristic of non-specific adsorption or partitioning. Intracellular membranes of the hepatocyte also bound [3H]cytochalasin B, those of the smooth endoplasmic reticulum to a greater extent than plasma membranes. [3H]Colchicine bound to plasma membranes in proportion to the amount of membrane and at a rate compatible with binding to tubulin. However, other properties of the binding including effects of temperature, drug concentration and antisera against tubulin were different from those of binding to tubulin. Hence, no evidence was obtained for association of microtubular elements with these membranes. Despite this there appeared to be an interdependence between microtubule and microfilament inhibitors: vinblastine sulfate stimulated [3H]cytochalasin B binding and cytochalasin B stimulated 3H colchicine binding. [3H]Colchicine also bound to intracellular membranes, especially smooth microsomes.     

Reconstitution of the glucose transporter from bovine heart

Reconstitution of the glucose transporter from heart should be useful as an assay in its purification and in the study of its regulation. We have prepared plasma membranes from bovine heart which display D-glucose reversible binding of cytochalasin B (33 pmol sites/mg protein; Kd = 0.2 muM). The membrane proteins were reconstituted into liposomes by the freeze-thaw procedure. Reconstituted liposomes showed D-glucose transport activity which was stereospecific, saturable and inhibited by cytochalasin B, phloretin, and mercuric chloride. Compared to membrane proteins reconstituted directly, proteins obtained by dispersal of the membranes with low concentrations of cholate or by cholate solubilization showed 1.2- or 2.3-fold higher specific activities for reconstituted transport, respectively. SDS-polyacrylamide gel electrophoresis followed by electrophoretic protein transfer and labeling with antisera prepared against the human erythrocyte transporter identified a single band of about 45 kDa in membranes from both dog and bovine hearts, a size similar to that reported for a number of other glucose transporters in various animals and tissues.     

Surface proteins of young and senescent cultured avian fibroblasts

Proliferation of senescent cultured chick fibroblasts is arrested at densities that are 3-4 fold lower than densities inhibiting growth of young cells. The effects of density and growth rate of young and aged cultures on the accessibility of their surface proteins to external iodination were studied. LETS glycoprotein and a protein of 110,000 daltons are the major iodinated proteins of resting, highly dense and of sparse young cells, respectively. By contrast, LETS is minimally exposed on undividing, relatively disperse old cells. Therefore, exposure of LETS is correlated with cell density rather than with growth rate.     

Effects of cocultivation with transformed cells on surface proteins of normal cells

Virally transformed fibroblasts do not have on their surface a major protein (large external transformation-sensitive, LETS) which is present in normal cells. Cocultivation of the transformation cells with normal cells whose surface proteins have been prelabelled induces an accelerated release of the LETS protein from the normal cells. We have investigated various conditions which affect this phenomenon. Our results show that alteration of cell surface proteins by cocultivation with the transformed cells is time and dose-dependent and requires cell contact. Serum was depleted at least 99% of plasminogen by affinity chromatography and used in the cocultivation experiments. It was found that activation of plasminogen was not required for the accelerated turnover of the LETS protein. Other diffusible proteases are also unlikely to be involved. The possibility that transformed cells have a membrane bound activity is discussed. The role of plasminogen activation was also tested for its relevance in transformation related proteolysis, growth and morphology of cells.     

Response of adrenal tumor cells to adrenocorticotropin: site of inhibition by cytochalasin B.

The ability of cytochalasin B to inhibit the steroidogenic response of mouse adrenal tumor cells (Y-1) to adrenocorticotropin (ACTH) was examined with two aims: to consider the specificity of the inhibitor and to determine at what point(s) in the steroidogenic pathway it acts. Cytochalasin B did not inhibit protein synthesis or transport of [3H]-cholesterol into the cells nor did it alter total cell concentration of ATP. Together with previous evidence, this suggests that the effects of cytochalasin observed are relatively specific in these cells. Cytochalasin inhibits the increase in conversion of [3H]cholesterol to 20alpha-[3H]dihydroprogesterone (20alpha-hydroxypregn-4-en-3-one: a major product of the steroid pathway in Y-1 cells) produced by ACTH but does not inhibit conversion of cholesterol to pregnenolone by mitochondrial and purified enzyme preparations from Y-1 cells and bovine adrenal, respectively. Cytochalasin does not inhibit the conversion of pregnenolone to 20alpha-dihydroprogesterone but was shown to inhibit increased transport of [3H]cholesterol to mitochondria resulting from the action of ACTH. These findings indicate that cytochalasin acts after cholesterol has entered the cells and before it is subjected to side-chain cleavage in mitochondria. In view of the known action of cytochalasin on microfilaments, it is proposed that these organelles are necessary for the transport of cholesterol to the mitochondrial cleavage enzyme and that at least one effect of ACTH (and cyclic AMP) is exerted upon this transport process. The specificity of the effects of cytochalasin is considered in relation to this conclusion.     

A model for the mode of action of cytochalasin B inhibition of D-glucose transport in the human erythrocyte.

By an optical method, cytochalasin B is shown to be a competitive inhibitor of D-glucose transport across the human erythrocyte membrane with Ki of 1.2 x 10(-7) M. A Drieding molecular model of cytochalasin B reveals an almost identical spatial distribution of four oxygen atoms to those found in the C1-conformation of beta-D-glucopyranose and implicated in hydrogen bonding to the carrier protein associated with D-glucose transport. The stereochemistry of this transport model is discussed. On the basis of the interoxygen distances found in cytochalasin B, hydrocortisone, prednisolone, corticosterone, and phenolphthalein are considered as analogues and are shown to be competitive inhibitors of D-glucose transport with Ki values of 2.2 x 10(-4) M, 3.0 x 10(-4) M, 4.0 x 10(-4) M, and 2.5 x 10(-5) M, respectively. These results are considered to be consistent with the proposed mode of action of cytochalasin B and also provide further support for the model of D-glucose stereospecifically hydrogen-bonded to a carrier protein.