Glycolysis and glucose uptake in intact outer segments isolated from bovine retinal rods

Glucose transport across the plasma membrane of isolated bovine rod outer segments (ROS) was measured by uptake of 14C-labeled 3-O-methylglucose and 2-deoxyglucose and was inferred from deenergization of ROS with 2-deoxyglucose. Glucose transport was mediated by a facilitated diffusion glucose transporter that equilibrated external and internal free hexose concentrations. Glucose transport in ROS displayed two components as judged from kinetic analysis of hexose equilibration and as judged from inhibition by cytochalasin B and phloretin. Transport under exchange conditions was considerably faster as compared with net hexose uptake, similar to that observed for the erythrocyte glucose transporter. Sensitivity to cytochalasin B and affinity to 3-O-methylglucose were similar to those observed for the hepatocyte glucose transporter. The cytochalasin-insensitive component appears unique to ROS and did not reflect leakage transport as judged from a comparison with L-glucose uptake. Glucose transport feeds glycolysis localized to ROS. We suggest that a major role for glycolysis in ROS is phosphorylation of GDP to GTP via pyruvate kinase and PEP, while phosphorylation of ADP to ATP can use the creatine kinase/phosphocreatine pathway as well.     

Kinetic characterization and radiation-target sizing of the glucose transporter in cardiac sarcolemmal vesicles

Stereospecific glucose transport was assayed and characterized in bovine cardiac sarcolemmal vesicles. Sarcolemmal vesicles were incubated with D-[3H]glucose or L-[3H]glucose at 25 degrees C. The reaction was terminated by rapid addition of 4 mM HgCl2 and vesicles were immediately collected on glass fiber filters for quantification of accumulated [3H]glucose. Non-specific diffusion of L-[3H]glucose was never more than 11% of total D-[3H]glucose transport into the vesicles. Stereospecific uptake of D-[3H]glucose reached a maximum level by 20 s. Cytochalasin B (50 microM) inhibited specific transport of D-[3H]glucose to the level of that for non-specific diffusion. The vesicles exhibited saturable transport (Km = 9.3 mM; Vmax = 2.6 nmol/mg per s) and the transporter turnover number was 197 glucose molecules per transporter per s. The molecular sizes of the cytochalasin B binding protein and the D-glucose transport protein in sarcolemmal vesicles were estimated by radiation inactivation. These values were 77 and 101 kDa, respectively, and by the Wilcoxen Rank Sum Test were not significantly different from each other.     

Distribution of the Glucose Transporter in the Mammalian Brain

We used [3H]cytochalasin B as a specific ligand to study the glucose transporter of the following tissue preparations: (a) microvessels derived from the cerebral cortex and cerebellum of the rat and pig, (b) particulate fractions of the cerebral cortex and cerebellum of the rat and pig, (c) lateral, third, and fourth ventricular choroid plexus of the pig, and (d) synaptosomes from the pig cerebral cortex. Specific, D-glucose-displaceable binding of [3H]cytochalasin B was present in all the preparations studied. This binding was saturable and displayed the kinetics of a single class of binding sites, similar to the glucose transporter found in other mammalian tissues. The density of the glucose transporter was much higher in cerebral and cerebellar microvessels and choroid plexus than either in crude particulate fractions of the cerebrum and cerebellum or in cerebral synaptosomes. These findings agree with the physiologic function of brain microvessels that transport glucose, not only for their own use, but also for the much greater mass of the entire brain. In the pig, the density of the glucose transporter in cerebral microvessels was significantly higher than in cerebellar microvessels. Irreversible photoaffinity labeling of the glucose transporter of synaptosomal membranes with [3H]cytochalasin B followed by solubilization and polyacrylamide gel electrophoresis demonstrated a single region of radioactivity that corresponded to a molecular mass of 60,000-64,000 daltons.     

Evidence that forskolin binds to the glucose transporter of human erythrocytes

Binding of [4-3H]cytochalasin B and [12-3H]forskolin to human erythrocyte membranes was measured by a centrifugation method. Glucose-displaceable binding of cytochalasin B was saturable, with KD = 0.11 microM, and maximum binding approximately 550 pmol/mg of protein. Forskolin inhibited the glucose-displaceable binding of cytochalasin B in an apparently competitive manner, with K1 = 3 microM. Glucose-displaceable binding of [12-3H]forskolin was also saturable, with KD = 2.6 microM and maximum binding approximately equal to 400 pmol/mg of protein. The following compounds inhibited binding of [12-3H]forskolin and [4-3H]cytochalasin B equivalently, with relative potencies parallel to their reported affinities for the glucose transport system: cytochalasins A and D, dihydrocytochalasin B, L-rhamnose, L-glucose, D-galactose, D-mannose, D-glucose, 2-deoxy-D-glucose, 3-O-methyl-D-glucose, phloretin, and phlorizin. A water-soluble derivative of forskolin, 7-hemisuccinyl-7-desacetylforskolin, displaced equivalent amounts of [4-3H]cytochalasin B or [12-3H]forskolin. Rabbit erythrocyte membranes, which are deficient in glucose transporter, did not bind either [4-3H]cytochalasin B or [12-3H]forskolin in a glucose-displaceable manner. These results indicate that forskolin, in concentrations routinely employed for stimulation of adenylate cyclase, binds to the glucose transporter. Endogenous ligands with similar specificities could be important modulators of cellular metabolism.     

Decrease in glucose transport activity of Friend erythroleukemia cell caused by dimethylsulfoxide, a differentiation-inducing reagent

A transport system for D-glucose was found in a Friend erythroleukemia cell line, T-3-C1-2-O and was characterized as a facilitated diffusion system. D-Glucose transport activity showed a half-saturation concentration of 2.2 mM and was inhibited by mercuric ions, cytochalasin B, phloretin, and stilbestrol, but was not strongly inhibited by phloridzin. Transport of 3-O-methyl-D-glucose was faster than D-glucose and the intracellular concentration of the sugar was found to reach the concentration in the assay medium. The treatment of cells with a differentiation-inducing reagent, dimethylsulfoxide(Me2SO), for 24 h caused a marked decrease in glucose transport activity due to a decrease in Vmax. In an induction-insensitive Friend cell line, T-3-K-1, D-glucose transport activity was low in untreated cells and Me2SO treatment did not cause a significant decrease in transport activity. The results obtained in this study indicate that the decrease in glucose transport activity is not due to the direct effect of Me2SO on transport activity, but is associated with the induction of differentiation. By immunoblotting cell lysates of T-3-C1-2-O cells using antibody to human erythrocyte glucose transporter, a single major band having a molecular weight of 52,000 was detected, which may be a glucose transporter in Friend cells.     

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.     

Molecular characteristics of rat brain glucose transporter: a novel species with Mr 45,000

The glucose transporter of rat brain was examined by the use of cytochalasin B, a potent inhibitor. The dissociation constants (Kd) of D-glucose-inhibitable cytochalasin B binding in various membrane fractions were about 100 nM. Solubilization and partial purification of glucose transporter were carried out by procedures of DE 52 column chromatography, Bio Gel HT column chromatography and Sepharose CL-6B column chromatography from postnuclear membrane fraction. Purified transporter, reconstituted in lipid vesicles, showed D-glucose-specific transport activity with a Michaelis constant (Km) of 7 mM. The molecular weight was estimated to be about 200K by gel filtration in the presence of 0.1% Triton X-100. The subunit molecular weight was estimated to be 45K by SDS-polyacrylamide gel electrophoresis after photoaffinity labeling using [3H]cytochalasin B as a covalent probe, indicating that rat brain glucose transporter is a tetramer.     

D-Glucose-sensitive and -insensitive cytochalasin B binding proteins from microvillous plasma membranes of human placenta. Identification of the D-glucose transporter

Cytochalasin B was found to bind to at least two distinct sites in human placental microvillous plasma membrane vesicles, one of which is likely to be intimately associated with the glucose transporter. These sites were distinguished by the specificity of agents able to displace bound cytochalasin B. [3H]Cytochalasin B was displaceable at one site by D-glucose but not by dihydrocytochalasin B; it was displaceable from the other by dihydrocytochalasin B but not by D-glucose. Some binding which could not be displaced by D-glucose + cytochalasin B binding site. Cytochalasin B can be photoincorporated into specific binding proteins by ultraviolet irradiation. D-Glucose specifically prevented such photoaffinity labeling of a microvillous protein component(s) of Mr = 60,000 +/- 2000 as determined by urea-sodium dodecyl sulfate acrylamide gel electrophoresis. This D-glucose-sensitive cytochalasin B binding site of the placenta is likely to be either the glucose transporter or be intimately associated with it. The molecular weight of the placental glucose transporter agrees well with the most widely accepted molecular weight for the human erythrocyte glucose transporter. Dihydrocytochalasin B prevented the photoincorporation of [3H]cytochalasin B into a polypeptide(s) of Mr = 53,000 +/- 2000. This component is probably not associated with placental glucose transport. This report presents the first identification of a sodium-independent glucose transporter from a normal human tissue other than the erythrocyte. It also presents the first molecular weight identification of a human glucose-insensitive high-affinity cytochalasin B binding protein.     

Transport of glucose by Bifidobacterium animalis subsp. lactis occurs via facilitated diffusion

Two strains of Bifidobacterium animalis subsp. lactis were indistinguishable by several nucleic acid-based techniques; however, the type strain DSMZ 10140 was glucose utilization positive, while RB 4825, an industrially employed strain, was unable to grow rapidly on glucose as the principal carbon source. This difference was attributed to the presence of a low-affinity facilitated-diffusion glucose transporter identified in DSMZ 10140 but lacking in RB 4825. Uptake of D-[U-(14)C]glucose in DSMZ 10140 was stimulated by monovalent cations (ammonium, sodium, potassium, and lithium) and inhibited by divalent cations (calcium and magnesium). When competitor carbohydrates were included in the uptake assays, stereospecific inhibition was exhibited, with greater competition by methyl-beta-glucoside than methyl-alpha-glucoside. Significant inhibition (>30%) was observed with phloretin, an inhibitor of facilitated diffusion of glucose, whereas there was no inhibition by sodium fluoride, iodoacetate, sodium arsenate, sodium azide, 2,4-dinitrophenol, monensin, or valinomycin, which typically reduce energy-driven transport. Based on kinetic analyses, the mean values for K(t) and V(max) were 14.8 +/- 3.4 mM D-glucose and 0.13 +/- 0.03 micromol glucose/min/mg cell protein, respectively. Glucose uptake by several glucose-utilizing commercial strains of B. animalis subsp. lactis was also inhibited by phloretin, indicating the presence of facilitated diffusion glucose transporters in those strains. Since DSMZ 10140 has been previously reported to lack a functional glucose phosphoenolpyruvate phosphotransferase system, the glucose transporter identified here is responsible for much of the organism's glucose uptake.     

Glycolytic enzymes and a GLUT-1 glucose transporter in the outer segments of rod and cone photoreceptor cells

The presence of glycolytic enzymes and a GLUT-1-type glucose transporter in rod and cone outer segments was determined by enzyme activity assays, glucose uptake measurements, Western blotting, and immunofluorescence microscopy. Enzyme activities of six glycolytic enzymes including hexokinase, phosphofructokinase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, and lactate dehydrogenase, were found to be present in purified rod outer segment (ROS) preparations. Immunofluorescence microscopy of bovine and chicken retina sections labeled with monoclonal antibodies against glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and lactate dehydrogenase have confirmed that these enzymes are present in rod and cone outer segments and not simply contaminants from the inner segments or other cells. Rod outer segments were also found to contain glucose transport activity as detected by 3-O-[14C]methylglucose uptake and exchange. The glucose transporter had a Km of 6.3 mM and a Vmax of 0.15 nmol of 3-O-methylglucose/s/mg of ROS membrane protein for net uptake and a Km of 29 mM and a Vmax of 1.06 nmol of 3-O-methylglucose/s/mg of ROS membrane protein for equilibrium exchange. These Km values for net uptake and equilibrium exchange are similar to values obtained for human red blood cells and are characteristic of GLUT-1-type glucose transporter. The transport was inhibited by both cytochalasin B and phloretin. Western blot analysis and immunofluorescence microscopy using type-specific glucose transporter antibodies indicated that both rod and cone outer segment plasma membranes have a GLUT-1 glucose transporter of Mr 45K as found in red blood cells and brain microsomal membranes. Solid-phase radioimmune competitive inhibition studies indicated that rod outer segment plasma membranes contained 15% the number of glucose transporters found in human red blood cell membranes and had an estimated density of 400 glucose transporter per micron2 of plasma membrane. These studies support the view that outer segments can generate energy in the form of ATP and GTP by anaerobic glycolysis to supply at least some of the energy requirements for phototransduction and other metabolic processes.     

Ascorbate uptake in normal and diabetic rat retina and retinal pigment epithelium

Oxidative stress is an important causative factor in the pathogenesis of diabetic retinopathy. Therefore, it becomes important to understand the mechanisms that help maintain appropriate levels of a small molecule antioxidant such as ascorbate in the retina. The outer blood-barrier which results from the tight junctions between the retinal pigment epithelial cells (RPE) restricts the flow of nutrients reaching the retina. In this study, we characterized the transport properties of carboxyl-(14)C ascorbate (AA) in normal rat retina and RPE, and compared them with those in streptozotocin-diabetic rats. Retina and RPE accumulated AA by a temperature-sensitive and energy-dependent kinetic mechanism with an apparent K(M) of 380 and 420 microM, respectively. Accumulation of AA was significantly reduced in a sodium-free medium. Although high glucose concentrations reduced AA uptake by 40%, this was not affected by cytochalasin B. The RPE and retina of diabetic rats presented lower levels of AA accumulation. These findings suggest the presence of the specific vitamin C transporter SVCT in retina and RPE, which may be involved in the manifestation of diabetic retinopathy.     

Basolateral glucose transport in distal segments of the dog nephron

The transport of glucose by canine thick ascending limbs (TAL) and inner medullary collecting ducts (IMCD) was studied using tubule suspensions and membrane vesicles. The uptake of D-[14C(U)]glucose by a suspension of intact TAL tubules was reduced largely by phloretin (Pt), moderately by phlorizin (Pz), and completely suppressed by a combination of both agents. A selective effect of Pz on the transport of [14C]alpha-methyl-D-glucoside, but not on 2-[3H]deoxyglucose, was also observed in TAL tubules. In contrast, glucose transport was unaffected by Pz but entirely suppressed by Pt alone in IMCD tubules. The metabolism of glucose was largely suppressed by Pt but unaffected by Pz in both types of tubules. Membrane vesicles were prepared from the red medulla and the white papilla or from TAL and IMCD tubules isolated from these tissues. Vesicle preparations from both tissues demonstrated a predominant carrier-mediated, sodium-independent, Pt- and cytochalasin B-sensitive glucose transport. Following purification of basolateral membrane on a Percoll gradient, the sodium-insensitive D-[14C(U)]glucose transport activity copurified with the activity of the basolateral marker Na(+)-K+ ATPase in both tissues. However, a small sodium-dependent and Pz-sensitive component of glucose transport was found in membrane vesicles prepared from the red medulla or from thick ascending limb tubules but not from the papilla nor collecting duct tubules. The kinetic analysis of the major sodium-independent processes showed that the affinity of the transporter for glucose was greater in collecting ducts (Km = 2.3 mM) than in thick ascending limbs (Km = 4.9 mM). We conclude that glucose gains access into the cells largely through a basolateral facilitated diffusion process in both segments. However a small sodium-glucose cotransport is also detected in membranes of TAL tubules. The transport of glucose presents an axial differentiation in the affinity of glucose transporters in the renal medulla, ensuring an adequate supply of glucose to the glycolytic inner medullary structures.     

Transport and metabolism of glucose by dissociated brain cells: Effects of trypsin

A study was carried out to determine the effect of trypsin on glucose transport into brain cells. Two suspensions of dissociated cells were prepared from the two brain hemispheres of adult rats—one using only mechanical means to dissociate the cells and one using trypsin. The use of trypsin for preparation of dissociated brain cells caused a marked reduction in the rate of transport of [1,2-³H]-2-deoxy-d-glucose compared to uptakes of this glucose analog by cells prepared without trypsin. Responses of the two cell preparations to inhibitors of glucose transport (cytochalasin B and phloretin) were similar. Rates of oxidation of [6-¹⁴C]glucose to¹⁴CO₂ by trypsin-treated cells were nearly double those in cells prepared without trypsin. Electron microscopic examination of the two preparations revealed much less preservation of structural integrity if trypsin was used to prepare the cells. The findings suggest that trypsin alters cell structure and affects receptor-regulated events in brain cells.     

Reconstitution of the glucose transporter from rat skeletal muscle

As a step in the purification and characterization of the glucose transporter from rat skeletal muscle, we have reconstituted glucose transport activity in liposomes. Plasma membranes were prepared from skeletal muscle which display D-glucose reversible binding of cytochalasin B (10 pmol sites/mg protein; KD = 0.3 microM). Older rats gave a slightly lower specific activity and much lower yield of sites per g muscle than young rats. Glucose transport activity was reconstituted into liposomes by the freeze-thaw procedure using either plasma membranes directly or cholate-extracted membrane proteins; the latter gave a 50% higher specific activity. The reconstituted transport activity was stereospecific, saturable, and inhibited by cytochalasin B, phloretin, and mercuric chloride. The optimum cholate concentration for extraction and reconstitution of transport activity was about 1.5%, and the highest specific activity of reconstituted transport was seen only at low ratios of protein to lipid in the reconstitution. Chromatography on agarose lentil lectin and agarose ethanethiol doubled both the specific activity of reconstituted transport and the fraction of glucose uptake which was stereospecific. In all of these respects the results were similar to our results with the bovine heart transporter (T. J. Wheeler and M. A. Hauck, Biochim. Biophys. Acta 818, 171-182 (1985)). Our findings suggest that further purification procedures developed for the heart transporter may be applicable to the skeletal muscle transporter as well.     

Is active glucose transport present in bovine ciliary body epithelium?

Hyperglycemia is a major risk factor for diabetic cataract formation. Effective regulation of glucose transport by the ciliary body epithelium (CBE) is pivotal to normal glycemic control in the anterior eye, which in turn affects the glucose level of the crystalline lens. The present study aimed to characterize the glucose transport mechanisms across the bovine blood-aqueous barrier (BAB) represented by the CBE. With an Ussing-type chamber, the glucose transport kinetics were measured and characterized in the presence and absence of various glucose transporter inhibitors. The saturation characteristics of the CBE to glucose were estimated from an Eadie-Hofstee plot. The mRNA expression of glucose transporters in specific regions of the bovine CBE was assessed using RT-PCR. The trans-CBE glucose flux was found to be sensitive to the glucose transporter inhibitors cytochalasin B, phloretin, and phlorizin. The transport system had a kinetic constant of 5.3 mM and a maximum velocity of 349.5 nmol.h(-1).cm(-2). Gene expression for GLUT1, GLUT3, GLUT4, GLUT5, and SGLT2 was observed in both the pars plana and pars plicata regions of the bovine CBE. This study demonstrates that glucose transport across the bovine CBE is primarily passive in nature. However, the novel findings of 1) the presence of a phlorizin-sensitive glucose flux and 2) gene expression for SGLT2 mean that a potential role for active glucose transport cannot be ruled out. The elucidation of the exact function of SGLT2 in the bovine CBE may shed important light on the glucose transport and physiology of the BAB and inform future studies of glycemic control in relation to diabetic cataract formation.     

Inhibition of transepithelial osmotic water flow by blockers of the glucose transporter

On the basis of evidence derived mostly from human erythrocytes, it has been suggested that water traverses cell membranes through membrane-spanning proteins such as the anion channel or the glucose transporter acting as water pores. However, specific inhibitors of such permeation processes have not been found to block water transport, and hence a precise identification of the water route has not been possible so far. We have investigated this issue by characterizing the osmotic flows across a fluid-transporting epithelium, the rabbit corneal endothelium. The rate of such flows was monitored continuously as a function of time. We confirmed prior findings of an inhibition by PCMBS on osmotic water flow, and lack of inhibition by DTNB and DIDS. On the other hand, we have found for the first time that several blockers of glucose facilitated diffusion, namely, phloretin (2 mM), phloridzin (2 mM), diallyldiethylstilbestrol (0.1 mM), cytochalasin B (20 micrograms/ml), and ethylidene-D-glucose (200 mM), all clearly inhibit osmotic flow. Our evidence is consistent with the hypothesis that both water and glucose may traverse these cell membranes through the same channel-like pathway contained in the glucose transporter membrane-spanning protein.     

D-Glucose uptake in fish hepatocytes: mediated by transporter in rainbow trout (Oncorhynchus mykiss), but only by diffusion in prespawning lamprey (Lampetra fluviatilis) and in RTH-149 cell line

The transport of D-glucose into rainbow trout (Oncorhynchus mykiss) and river lamprey (Lampetra fluviatilis) hepatocytes, as well as into rainbow trout hepatoblastoma cell line RTH-149 was studied using tracer methods. The half-time for D-glucose equilibration was 15 s for rainbow trout. The half-times for the non-metabolizable D-glucose analog, 3-O-methyl-D-glucose equilibration were 8 s, 37 s and 38 s for rainbow trout, lamprey and RTH-149 cells, respectively. The 3-O-methyl-D-glucose was taken up by rainbow trout hepatocytes by facilitated diffusion in addition to simple diffusion. The uptake showed saturation kinetics with the K(m) of 37 mM and V(max) of 62 mmol kg(-1) cells min(-1). The uptake was sensitive to phloretin and cytochalasin B, but not affected by ouabain. The 3-O-methyl-D-glucose uptake by lamprey hepatocytes and RTH-149 cells showed no indication of saturation up to 160 mM, and was not affected by phloretin, cytochalasin B or ouabain, which suggests the mode of transport to be by passive diffusion. However, immunocytochemical stainings revealed the existence of mammalian type GLUT1 and GLUT2 transporters in all cells studied. The lack of a functioning carrier-mediated glucose uptake in lamprey hepatocytes might be due to its physiological state (prespawning starvation). The minor 3-O-methyl-D-glucose uptake into RTH-149 cells compared to freshly isolated rainbow trout hepatocytes might reflect low metabolic activity of the cell lines. Under the conditions applied the RTH-149 cell line is no suitable in vitro model for glucose transport in fish cells.     

The red blood cell glucose transporter presents multiple, nucleotide-sensitive sugar exit sites.

At any instant, the human erythrocyte sugar transporter presents at least one sugar export site but multiple sugar import sites. The present study asks whether the transporter also presents more than one sugar exit site. We approached this question by analysis of binding of [3H]cytochalasin B (an export conformer ligand) to the human erythrocyte sugar transporter and by analysis of cytochalasin B modulation of human red blood cell sugar uptake. Phloretin-inhibitable cytochalasin B binding to human red blood cells, to human red blood cell integral membrane proteins, and to purified human red blood cell glucose transport protein (GluT1) displays positive cooperativity at very low cytochalasin B levels. Cooperativity between sites and K(d(app)) for cytochalasin B binding are reduced in the presence of intracellular ATP. Red cell sugar uptake at subsaturating sugar levels is inhibited by high concentrations of cytochalasin B but is stimulated by lower (<20 nM) concentrations. Increasing concentrations of the e1 ligand forskolin also first stimulate then inhibit sugar uptake. Cytochalasin D (a cytochalasin B analogue that does not interact with GluT1) is without effect on sugar transport over the same concentration range. Cytochalasin B and ATP binding are synergistic. ATP (but not AMP) enhances [3H]cytochalasin B photoincorporation into GluT1 while cytochalasin B (but not cytochalasin D) enhances [gamma-32P]azidoATP photoincorporation into GluT1. We propose that the red blood cell glucose transporter is a cooperative tetramer of GluT1 proteins in which each protein presents a translocation pathway that alternates between uptake (e2) and export (e1) states but where, at any instant, two subunits must present uptake (e2) and two subunits must present exit (e1) states.     

Polarity of transport of 2-deoxy-D-glucose and D-glucose by cultured renal epithelia (LLC-PK1).

At least two types of glucose transporter exist in cultured renal epithelial cells, a Na(+)-glucose cotransporter (SGLT), capable of interacting with D-glucose but not 2-deoxy-D-glucose (2dglc) and a facilitated transporter (GLUT) capable of interacting with both D-glucose and 2dglc. In order to examine the polarity of transport in cultured renal epithelia, 2dglc and D-glucose uptakes were measured in confluent cultures of LLC-PK1 cells grown on collagen-coated filters that permitted access of medium to both sides of the monolayer. The rates of basolateral uptake of both 1 mM glucose (Km 3.6 mM) and 1 mM 2dglc (Km 1.5 mM) were greater than apical uptake rates and the (apical-to-basolateral)/(basolateral-to-apical) flux ratio was high for glucose (9.4) and low for 2dglc (0.8), thus, confirming the lack of interaction of 2dglc with the apical SGLT. Specific glucose transport inhibitor studies using phlorizin, phloretin and cytochalasin B confirmed the polarised distribution of SGLT and GLUT in LLC-PK1 cells. Basolateral sugar uptake could be altered by addition of insulin (1 mU/ml) which increased 2dglc uptake by 72% and glucose uptake by 50% and by addition of 20 mM glucose to the medium during cell culture which decreased 2dglc uptake capacity at confluence by 30%. During growth to confluence, 2dglc uptake increased to a maximum, then decreased at the time of confluence, coincident with a rise in uptake capacity for alpha-methyl-D-glucoside, a hexose that interacts only with the apical SGLT. It was concluded that the non-metabolisable sugar 2dglc was a useful, specific probe for GLUT in LLC-PK1 cells and that GLUT was localised at the basolateral membrane after confluence.     

Derivatization of the human erythrocyte glucose transporter using a novel forskolin photoaffinity label

An iodinated photoaffinity label for the glucose transporter, 3-iodo-4-azidophenethylamido-7-O-succinyldeacetyl-forskolin (IAPS-forskolin), has been synthesized, purified, and characterized. The I50 for inhibition of 3-O-methylglucose transport in red blood cells by IAPS-forskolin was found to be 0.05 microM. The carrier free radioiodinated label is a highly specific photoaffinity label for the human erythrocyte glucose transporter. Photolysis of erythrocyte membranes (ghosts) and purified glucose transporter preparations with 1-2 nM [125I]IAPS-forskolin and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed specific derivatization of a broad band with an apparent molecular mass of 40-70 kDa. Photoincorporation into erythrocyte membranes using 2 nM [125I]IAPS-forskolin was protected with D-glucose (I50 400 mM), cytochalasin B (I50 0.5 microM), and forskolin (I50 10 microM). No protection was observed with L-glucose (600 mM). Endo-beta-galactosidase digestion of [125I] IAPS-forskolin-labeled ghosts and purified transporter resulted in a dramatic sharpening of the specifically radiolabeled transporter to 40 kDa. Trypsinization of [125I]IAPS-forskolin-labeled ghosts and purified transporter reduced the specifically radiolabeled transporter to a sharp peak at 18 kDa. [125I]IAPS-forskolin will be a useful tool to study the structural aspects of the glucose transporter.