Stereospecific Uptake Of D-glucose Research Articles

Influence of organic and inorganic arsenicals on glucose uptake in Madin-Darby canine kidney (MDCK) cells.

The effect of organic (oxophenylarsine; PhAsO) and inorganic (arsenite) arsenicals on the availability of glucose to Madin-Darby canine kidney (MDCK) cells was investigated. The MDCK cells revealed stereospecific D-glucose uptake which was inhibited by both arsenicals in a time- and concentration-dependent manner. After 10 min (37 degrees C), the effects on D-glucose and 2-deoxy-D-glucose accumulation were analogous, suggesting an impaired hexose uptake. With arsenite, 0.5-1 mmol dm-3 were required for half-maximum inhibition (IC50), whereas PhAsO inhibited glucose uptake in the micromolar range (IC50 5-30 mumol dm-3). Under these conditions neither cell morphology nor cellular viability was affected. After 60 min, however, the inhibition of glucose utilization was paralleled by the formation of blebs, detachment of the monolayer and a loss of cellular viability as confirmed by dye exclusion, lactate dehydrogenase and potassium release. It is concluded that inhibition of glucose uptake may contribute to the acute toxicity, especially of organic arsenicals, by further aggravating the depletion of intracellular carbohydrates.

Direct interaction of phenylarsine oxide with hexose transporters in isolated rat adipocytes

It has previously been shown that phenylarsine oxide (PhAsO), an inhibitor of protein internalization, also inhibits stereospecific uptake of d-glucose and 2-deoxyglucose in both basal and insulin-stimulated rat adipocytes. This inhibition of hexose uptake was found to be dose-dependent. PhAsO rapidly inhibited sugar transport into insulin-stimulated adipocytes, but at low concentrations inhibition was transient. Low doses of PhAsO (1 μM) transiently inhibit stereospecific hexose uptake and near total (approx. 90%) recovery of transport activity occurs within 20 min. Interestingly, once recovered, the adipocytes can again undergo rapid inhibition and recovery of transport activity upon further treatment with PhAsO (1 μM). In addition, PhAsO is shown to inhibit cytochalasin B binding to plasma membranes from insulin-stimulated adipocytes in a concentration-dependent manner which parallels the dose-response inhibition of hexone transport by PhAsO. The data presented suggest a direct interaction between the d-glucose transporter and PhAsO, resulting in inhibition of transport. The results are consistent with the current recruitment hypothesis of insulin activation of sugar transport and indicate that a considerable reserve of intracellular glucose carriers exists within fat cells.

The action of phenylarsine oxide on the stereospecific uptake of D-glucose in basal and insulin-stimulated rat adipocytes

Phenylarsine oxide (PAO) has been used to inhibit the stereospecific uptake of D-glucose in basal and insulin-stimulated rat adipocytes. The inhibition is dose dependent and is partially reversed by dithiothreitol. The results are consistent with a direct interaction between the glucose transporter and PAO. By manipulating the sequence of exposure of cells to PAO and insulin it is possible to differentiate between the effects of PAO on transport into cells with receptor-rich and transporter-rich plasma membranes. PAO rapidly inhibits transport in insulin-stimulated adipocytes but at low concentrations inhibition is transient and recovery of stereospecific uptake takes place after approx. 20 min. The results can be interpreted in terms of the recruitment mechanism of insulin stimulation of transport and demonstrate that a relatively large intracellular pool of transporters exists after insulin stimulation. It also follows that sulphydryl groups probably play a critical role in the mechanism of glucose uptake.

The effect of membrane composition and alcohols on the insulin-sensitive reconstituted monosaccharide-transport system of rat adipocyte plasma membranes.

The monosaccharide transporter from the plasma membranes of rat adipocytes and insulin-stimulated adipocytes has been reconstituted in sonicated liposomes. The stereospecific D-glucose uptake by liposomes made from a range of phospholipids and incorporating fatty acids has been investigated. D-Glucose uptake is correlated with an increase in lipid fluidity as a consequence of the addition of fluidizing fatty acids, changes in phospholipid acyl chain length and temperature. Benzyl alcohol and ethyl alcohol, which are generally considered to increase bilayer fluidity, decrease stereo-specific D-glucose uptake in both whole adipocytes and reconstituted liposomes. It is suggested that, although these alcohols may affect D-glucose transport by lipid-mediated fluidity changes, they also interact directly with the transporter resulting in inhibition of transport.

The glucose transport system of muscle plasma membranes: Characterization by means of [ 3H]cytochalasin B binding

A membrane-rich preparation was isolated from adult rat skeletal muscle in low salt media and further fractionated in sucrose gradients. Fraction F2, with a relative density of 1.092–1.119, consisted of sealed membrane vesicles which were enriched in plasma membrane markers. These vesicles were capable of stereospecific d-glucose uptake which was sensitive to cytochalasin B (CB). The membranes were also enriched in high affinity [ 3H]CB binding activity ( K d of 0.28 μ m). [ 3H]CB binding to the glucose carrier of these plasma membranes, estimated as the fraction of binding protectable by d-glucose, ranged between 2.5 and 7.4 pmol/mg protein in several membrane preparations. The amount of [ 3H]CB binding to muscle membranes from newborn and adult rats was not markedly different. Trypsin, at low concentrations, altered the molecular weight of several membrane components, without affecting [ 3H]CB binding. Higher concentrations of trypsin abolished [ 3H]CB binding. Both 2,4-dinitrofluorobenzene (0.1 m m) and N-ethylmaleimide (15 m m) inhibited [ 3H]CB binding; inhibition by these reagents was prevented by inclusion of micromolar concentrations of CB in the reaction mixture. Several procedures that extracted specific proteins enriched the d-glucose-sensitive [ 3H]CB binding to the protein-depleted membranes. Antibody raised against the glucose carrier of human red cell membranes cross-reacted with a polypeptide of M r about 45K of muscle membranes which might represent the glucose carrier.

In cultured chick embryo fibroblasts the hexose transport components are not the 75 000 and 95 000 dalton polypeptides synthesized following glucose deprivation

Glucose deprivation of chick embryo fibroblasts results in a cycloheximide-sensitive stimulation of hexose transport and an increase in the levels of glucose-regulated polypeptides of molecular weights 75 000 and 95 000. The relationship between these two phenomena is evaluated in this study. The glucose deprivation-induced stimulation of hexose transport was observed to occur in two phases: a rapid (complete by 15 min) cycloheximide-insensitive increase of 50-100% and a slower (observable by 6 h) cycloheximide-sensitive increase in transport to about five times the basal level. The time course of the latter increase preceded that of the appearance of the 75 000 and 95 000 dalton polypeptides; by the time that increases in the levels of these polypeptides were observed, the hexose uptake rates had almost reached their maximum value. Upon cellular fractionation, the greatest enrichment of the 75 000 and 95 000 dalton polypeptides was observed in the endoplasmic reticulum fraction, which was devoid of vesicular stereospecific D-glucose uptake activity. The plasma membrane fraction was enriched in stereospecific D-glucose uptake activity. The plasma membrane fraction was enriched in stereospecific D-glucose uptake activity but not in the 75 000 and 95 000 dalton polypeptides. The glucose deprivation-induced increase in hexose uptake was not prevented by tunicamycin, although this inhibitor of protein glycosylation decreased the hexose uptake of glucose-fed cells by 80% after 24 h. However, under these latter conditions an increase in the levels of the 75 000 and 95 000 dalton polypeptides was observed. On the basis of this data, we conclude that the polypeptides of molecular weights 75 000 and 95 000 are not involved in glucose transport.

Stereospecific d-glucose transport in mixed membrane and plasma membrane vesicles derived from cultured chick embryo fibroblasts

Mixed membrane vesicles prepared from cultured chick embryo fibroblasts possess a stereospecific d-glucose transport system, the properties of which are identical to those of the system in intact cells. Uptake of d-glucose proceeds without chemical alteration. The rate of stereospecific uptake of d-glucose into the mixed vesicles is 70% greater than that of the homogenate and uptake is directly proportional to membrane protein concentration. Stereospecific d-glucose uptake appears linear for 0.3 min, reaches a maximum at 2–5 min, and declines to zero by 5 h as l-glucose enters the vesicles. Uptake is osmotically sensitive and inhibited by cytochalasin B ( K i = 0.13 μM) and the structural analogues of d-glucose : d-mannose, 2-deoxy- d-glucose, 3- O- methyl- d- glucose , d-galactose and maltose, but not by sucrose or l-glucose. Uphill counterflow can be demonstrated and the apparent activation energy displays a transition from 47.7 kcal/mol below 11°C to 18.1 kcal/mol above 11°C. Stereo-specific uptake rates of mixed vesicles prepared from Rous sarcoma virus-transformed cells are increased 30% over control values, and are increased 66% in vesicles derived from cells incubated for 24 h in glucose-free medium. Plasma membrane vesicles prepared from these cells by a dextran cushion centrifugation procedure display a 9-fold increase in the specific activity of stereospecific d-glucose uptake relative to the homogenate. Extraction of these membranes with dimethylmaleic anhydride (5 mg/mg protein) results in substantial or complete removal of major polypeptides of molecular weight 40 000, 55 000, 75 000, 78 000 and 200 000 with no loss in total uptake activity. Following extraction, major polypeptides of molecular weight 28 000, 33 000 and 68 000 remain in the membrane residue.

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.

Specific d-glucose transport in sarcolemma vesicles

The sarcolemmal fraction prepared from rat skeletal muscle consists of osmotically active vesicles that accumulate d-glucose in preference to l-glucose, apparently by facilitated diffusion into intravesicular space. Stereospecific d-glucose uptake by these vesicles is a saturable process, inhibited by phloridzin, by cytochalasin B, and by certain sugars, and enhanced by counterflow. An additional leak pathway permits entry of both d- and l-glucose into the vesicles. Stereospecific d-glucose transport by sarcolemmal vesicles is enhanced to a small extent by insulin, provided the hormone is administered prior to cell disruption. In membranes prepared from insulin-pretreated muscle, Ca 2+ produces a small further enhancement. Local anesthetics preferentially inhibit stereospecific d-glucose transport. Apparent uptake of both d- and l-glucose is greater when vesicles are suspended in salt solutions rather than sucrose, an effect attributed to increased functional vesicular volume.

Preferential uptake of D-glucose by plasma membranes isolated from human adipose tissue.

A sensitive method for the measurement of the stereospecific uptake of D-glucose by plasma membranes isolated from human adipose tissue has been developed. The method is based on the difference in uptake of L-[14C]glucose and D-[3H]glucose as measured by the retention of radioactivity by the membrane preparation collected on Millipore filters. This D-glucose-uptake activity was reversible and did not involve any chemical alteration of the sugar. All uptake activity was lost upon boiling the membrane preparation for 5-10 min. All of the hydroxyl groups of D-glucose appear to be involved in a concerted fashion in the uptake reaction. The D-glucose-uptake activity was shown to be closely associated with glucose transport in adipose cells, since it exhibited the following properties characteristic of this carrier-mediated transport system. (a) The uptake was specific for the D-isomer of glucose. (b) Saturation of D-glucose uptake occurred with increasing concentrations of D-glucose, (c) The uptake activity was inhibited by N-ethylamleimide and phloretin, two reagents previously reported to inhibit D-glucose transport. We conclude that plasma membranes isolated from human adipose tissue retain the glucose transport activity of the intact cells and can be used in subsequent attempts at the isolation and characterization of this transport system.

Partial puridication of a membrane protein from human erythrocytes involved in glucose transport.

In an attempt to determine which membrane proteins are essential to the stereospecific uptake of D-glucose, isolated human erythrocyte membranes were exposed to a variety of reagents capable of selectively extracting various membrane proteins. These reagents included EDTA, lithium 3,5-diiodosalicylate, sodium iodide, and 2,3-dimethylmaleic anhydride. Selective elution of spectrin and Components 2.1, 2.2, 2.3, 4.1, 4.2, 5, and 6 representing 65% of the ghost protein has no effect on the uptake of D-glucose. All of the sugar transport proteins are associated with a membrane residue consisting of the proteins of Bands 3, 4.5, and 7, the periodic acid-Schiff-sensitive glycoproteins, and ghost phospholipids. Specific cross-linking of the proteins of Band 3 of ghosts by the catalyzed oxidation of intrinsic sulfhydryl groups with the o-phenanthroline-cupric ion complex inhibits D-glucose uptake and alters the relative electrophoretic mobility of Band 3 proteins in sodium dodecyl sulfate-polyacrylamide-agarose gels. This uptake activity and the relative mobility of Band 3 proteins are recovered upon reversal of the cross-linking reaction by reduction with 2-mercaptoethanol. These results and other observations indicate that the D-glucose transport protein is an intrinsic component of the hydrophobic structure of the erythrocyte membrane and may be associated with the proteins of Band 3 which are glycoproteins spanning the membrane bilayer. It is proposed that D-glucose transport occurs through a water-filled channel formed by specific subunit aggregates of the transport proteins in the erythrocyte membrane rather than by rotation of the protein within the plane of the membrane.

Studies on the mechanism and reversal of the phospholipase-A2 inactivation of D-glucose uptake by isolated human erythrocyte membranes.

The mechanism underlying the inactivation of the stereospecific uptake of D-glucose by isolated human-erythrocyte membranes following digestion with phospholipase A2 (Kahlenberg, A. &Banjo, B. (1972) J. Biol. Chem 247, 1156–1160) was investigated. This inactivation was not accompanied by any significant change in the uptake of L-glucose. The decrease in D-glucose uptake following limited (25–30%) cleavage of membrane phospholipids by phospholipase A2 was characterized by a twofold increase in the apparent dissociation constant of the D-glucose–membrane complex and a 34% decrease in the membrane's maximum capacity for D-glucose uptake. These effects of phospholipase A2 were completely reversed upon removal of the membrane-bound phospholipid byproducts (fatty acids and lysophospholipids) by washing the membranes with defatted bovine-serum albumin. Oleic acid and various lysophosphatides added to albumin-washed, phospholipase A2-treated membranes in amounts formed by the enzyme treatment produced negligible inhibition of D-glucose uptake. With more extensive phospholipase A2 digestion of membrane phospholipids, defatted bovine-serum albumin did not restore D-glucose uptake despite the removal of the phospholipid by-products formed.In addition to the inactivation of D-glucose uptake, limited enzyme treatment transforms the appearance of the membranes collected by centrifugation from opaque white to transparent and gelatinous. Both of these effects of phospholipase A2 are completely reversed upon incubation of the membranes at pH 5.5 for 2 h at 37 °C without loss of any of the membrane lysophosphatides and fatty acids formed by the enzyme treatment. It is suggested that this pH- and temperature-dependent restoration of D-glucose uptake is due to a conformational change resulting in the relocation of the membrane D-glucose-binding sites into a functional environment.These results indicate that the inactivation of D-glucose uptake by phospholipase A2, which was not accompanied by any change in L-glucose uptake, occurs by two different mechanisms. With limited hydrolysis of membrane phospholipids, one or both of the resulting phospholipid by-products reversibly inhibit the uptake of D-glucose by decreasing the affinity of the membrane for D-glucose and by masking a portion of the total available D-glucose-binding sites. However, upon extensive cleavage of phospholipids in the hydrophobic region of the membrane, there is an apparently irreversible disorganization of the membrane D-glucose-binding component. This might be due to destruction of vital phospholipids and/or a disturbance of the interactions between the lipid and protein components of the membrane.

Further Studies on the Previously Proposed Saturable High Affinity Site for d-Glucose in Renal Brush Border Membrane Preparations

Abstract Measurements of the stereospecific uptake of micromolar concentrations of d-glucose by rabbit renal brush border preparations suggested saturation at a affinity only when incubations at 20° were prolonged, but not when the incubation period was ½ min. This prompted a renewed effort to ascertain whether the apparent saturability at the longer incubation periods was due to bacterial contamination. The following precautionary measures were taken: (a) brush border suspensions were cultured for 24 to 72 hours at 37°, and little or no bacterial growth was found; (b) all incubations were carried out in the presence of penicillin and streptomycin; and (c) preincubation of the membrane suspensions at 37° decreased the uptake of d-glucose. Although these observations seemed to argue counter to bacterial contamination as a causative factor, subsequent studies revealed the presence in reagent solutions of a temperature (37°) sensitive, antibiotic resistant, bacterium of the genus Pseudomonas. These bacteria, isolated from the reagent solutions, cultured at 20°, and incubated at 20° with glucose showed a stereospecific uptake of d-glucose, with saturation kinetics at micromolar concentrations and sensitivity to preincubation at 37°. With the use of essentially bacteria-free brush border preparations and reagent solutions, saturation was not evident in the high affinity concentration range of 0.67 to 50 µm d-glucose. It is suggested that there is now no convincing evidence for the presence of the saturable high affinity site for d-glucose in renal brush borders and that only the Na+-dependent transport of the sugar saturable at higher concentrations is indicated. It is suggested, additionally, that the high affinity binding of d-glucose to intestinal brush border preparations, reported by others, warrants re-examination. Essentially, sterile renal brush borders bind phloridzin at a high affinity site, with a Kphloridzin of 7 µm, confirming reports of this site with other renal preparations.

D-Glucose Uptake by Isolated Human Erythrocyte Membranes versusd-Glucose Transport by Human Erythrocytes

Abstract The effects of proteolytic and phospholipase A2 digestion on the transport of d-glucose by intact human erythrocytes and on the uptake of d-glucose by isolated erythrocyte membranes have been studied. This stereospecific uptake of d-glucose was previously shown to be compatible with the over-all monosaccharide transport system in erythrocytes and evidence was presented that it represented the binding of d-glucose to a specific site on the erythrocyte membrane (Kahlenberg, A., Urman, B., and Dolansky, D. (1971) Biochemistry 10, 3154–3162. In the absence of hemolysis, no change is observed in the transport of d-glucose by intact erythrocytes exposed to the above enzymes or in the uptake of d-glucose by membranes isolated from enzyme-treated cells. In contrast, limited proteolytic digestion of isolated erythrocyte membranes, under conditions identical with those for the digestion of intact cells, results in a 3- to 5-fold increase in d-glucose uptake and a 2-fold increase in the apparent dissociation constant for the d-glucose-membrane complex. This increase in the uptake of d-glucose is different from the effects which result from the digestion of isolated membranes with phospholipase A2 (decreased uptake of d-glucose) or neuraminidase (no change in d-glucose uptake). More drastic tryptic digestion leads to a loss of the stimulated d-glucose uptake with no further change in the affinity of the membrane for the sugar. Pronase hydrolysis of 20 to 30% of the proteins of isolated membranes results in the destruction of 50 to 70% of the d-glucose uptake. These results indicate that the transport system in erythrocytes involves membrane components not normally accessible to proteolytic and phospholipase A2 digestion. Isolation of the erythrocyte membrane renders the d-glucose uptake activity of these membrane components susceptible to enzymatic modification. The evidence suggests that the increase in maximum d-glucose uptake resulting from limited proteolytic digestion of isolated membranes represents exposure of latent d-glucose binding sites which are masked by a protease-sensitive covering on the surface of the isolated membrane.

Involvement of Phospholipids in the d-Glucose Uptake Activity of Isolated Human Erythrocyte Membranes

Abstract A method for measuring the stereospecific uptake of d-glucose by isolated human erythrocyte membranes has recently been developed in our laboratory (Kahlenberg, A., Urman, B., and Dolansky, D. (1971) Biochemistry, 10,3154). Data obtained with this method were interpreted to suggest that the uptake activity measured represented the binding of d-glucose to a specific site on the erythrocyte membrane. To investigate the possible role of phospholipids in this d-glucose uptake activity, the phospholipid structure of erythrocyte membranes was modified by treatment with phospholipase A2, C, or D, and the effect of each modification on their d-glucose uptake activity and phospholipid composition was determined. Phospholipase A2 treatment produced a maximum decrease in uptake activity of 75% in association with the hydrolysis of approximately 74% of the total available β-fatty acid ester linkages of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine; the maximum hydrolysis of these phospholipids obtainable was 91%. In contrast, hydrolysis of approximately 64% and 46% of the membrane phospholipid phosphodiester linkages by phospholipase C and D, respectively, resulted in only a 25% decrease in d-glucose uptake activity. Likewise a greater decrease in the activity of the erythrocyte membrane (sodium plus potassium)-activated adenosine triphosphatase was observed after phospholipase A2 than after phospholipase C or D treatment. Acetylcholinesterase activity of the membrane was not affected by any of these phospholipolytic enzymes. The above results suggest that perturbation of phospholipid hydrophobic groups in the interior of the membrane, proximal or hydrophobically bound to membrane proteins, causes a decrease in the d-glucose uptake activity of these proteins.