3-O-methyl-D-glucose uptake in isolated bovine adrenal chromaffin cells

The characteristics and regulatory nature of sugar transport in freshly isolated bovine adrenal chromaffin cells were investigated. Transport was measured by following the cell/medium distribution of non-metabolizable glucose analogue, 3-O-methyl-D-glucose. The uptake of 3-O-methyl-D-glucose was was mediated by a saturable transport system with a Km of 8.2 mM and a Vmax of 0.69 nmol/mg protein per min. Basal 3-O-methyl-D-glucose transport was competitively inhibited by D-glucose and a countertransport effect was demonstrated. Cytochalasin B and phloretin, which are specific inhibitors of carrier-mediated glucose transport, significantly decreased basal 3-O-methyl-D-glucose uptake. Basal transport was stimulated by 50 mU/ml insulin, an effect associated with an increase in Vmax. The stimulatory effect of insulin was depressed in medium lacking external Ca2+, or containing the Ca2+-antagonistic ion, La3+, or the Ca2+ channel blocker, methoxyverapamil (D-600). The data suggest that the uptake of 3-O-methyl-D-glucose in freshly isolated bovine adrenal chromaffin cells is mediated by a specific facilitated diffusion mechanism, and is subject to regulation by insulin, thus resembling sugar transport in muscle. In addition, the insulin effect appears to depend on the presence of extracellular Ca2+.     

Studies on the persistence of enhanced monosaccharide transport in rat skeletal muscle following cessation of the initial stimulus

The activity of the facilitated diffusion system for monosaccharides remains enhanced in soleus and extensor digitorum longus muscles after a period of contractile activity but not in vas deferens smooth muscle. This memory effect could also be demonstrated in the skeletal muscle after exposure to a hyperosmolar buffer. Extracellular calcium ions did not influence the distribution of 3-O-methyl-D-glucose after contractile activity. The most likely explanation for the memory effect is a change in the physical nature of the plasma membrane rather than an alteration in the distribution of calcium ions.     

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.     

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.     

Action of insulin on the transport of 3-O-methylglucose in frog sartorius muscles

The initial rate of entry of 3-O-methyl-D-glucose into isolated frog sartorius muscles was measured at various concentrations of substrate, at 0, 9, 19, or 29 degrees C, after prior incubation at 19 degrees C with a maximally stimulating concentration of insulin. Control muscles were treated similarly, except for the omission of insulin. A saturable transport system provided for most of the entry of 3-O-methylglucose into muscle cells, but a small amount of penetration occurred by a nonsaturable route. The major effect of insulin was to produce a large increase in activity of the suturable system. The Vmax of entry increased, but there was no significant change in the apparent Km. The ratio of insulin-stimulated to basal Vmax was 10 when transport was measured at 29 degrees C but was 22 at 0 degrees C. These findings support the hypothesis that, although a large part of the effect of insulin on sugar transport can be accounted for by an increase in the number of functional transporters in the plasma membrane, there is a separate hormonal effect that permits a relatively greater activity of transporters at lower temperatures, compared with control rates. An additional effect of insulin was to produce a small but definite increase in the entry of sugar by the nonsaturable transport system.     

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.     

Utilization and transport of glucose in Olea Europaea cell suspensions

Cell suspensions of Olea europaea var. Galega Vulgar grown in batch culture with 0.5% (w/v) glucose were able to transport D-[(14)C]glucose according to Michaelis-Menten kinetics associated with a first-order kinetics. The monosaccharide carrier exhibited high affinity (K(m) approximately 50 micro M) and was able to transport D-glucose, D-fructose, D-galactose, D-xylose, 2-deoxy-D-glucose and 3-O-methyl-D-glucose, but not D-arabinose, D-mannitol or L-glucose. D-[(14)C]glucose uptake was associated with proton uptake, which also followed Michaelis-Menten kinetics. The transport of 3-O-methyl-D-glucose was accumulative (40-fold, at pH 5.0) and the protonophore carbonyl cyanide m-chlorophenylhydrazone strongly inhibited sugar accumulation. The results were consistent with the involvement of a monosaccharide: proton symporter with a stoichiometry of 1 : 1. When cells were grown with 3% (w/v) glucose, the uptake of D-[(14)C]glucose followed first-order kinetics and monosaccharide:proton symporter activity was not detected. The value obtained for the permeability coefficient of hexoses in O. europaea cells supported the hypothesis that the first-order kinetics observed in 0.5% and 3% sugar-grown cells was produced exclusively by passive diffusion of the sugar. The results indicate that in O. europaea cells sugar levels have a regulatory effect on sugar transport, because the activity for monosaccharide transport was repressed by high sugar concentrations.     

Regulation of 3-O-methyl-D-glucose uptake in isolated bovine adrenal chromaffin cells

We showed earlier that insulin stimulated sugar transport in adrenal chromaffin cells (Bigornia, L. and Bihler, I. Biochim. Biophys. Acta 885, 335-344). Transport regulation and its Ca2+ -dependence was further investigated in isolated bovine adrenal chromaffin cells, serving as a model of a homogeneous neuronal cell population. Uptake of the nonmetabolizable glucose analogue, 3-O-methyl-D-glucose was stimulated by hyperosmolar medium, and this effect was abolished in the absence of external Ca2+, or depressed in the presence of La3+ or the slow Ca2+ channel blocker methoxyverapamil. Basal transport was also stimulated by factors (acetylcholine, carbamylcholine, low-Na+ medium), which cause Ca2+ -dependent catecholamine release, and these effects were abolished in Ca2+ -free medium. In addition insulin, acetylcholine, hyperosmolar and low-Na+ medium significantly increased 45Ca uptake. Thus, glucose transport in adrenal chromaffin cells was stimulated by insulin and hyperosmolarity in a Ca2+ -dependent manner, as in muscle. Sensitivity to secretory stimuli, a regulatory feature perhaps characteristic of this cell type, was also demonstrated. In contrast to muscle, sugar transport was not affected by Na+ -pump inhibition, metabolic inhibitors or the Na+ ionophore monensin, suggesting that Ca2+ influx by Na+/Ca2+ exchange does not play a significant role in the activation of sugar transport in chromaffin cells.     

Direct measurements of sugar uptake in small and large adipocytes from young and adult rats

Measurements of basal and insulin-stimulated uptake of D-glucose, 2-deoxy-D-glucose and 3-O-methyl-D-glucose were determined in isolated fat cells from young and adult rats by an oil-centrifugation technique. At low sugar concentrations, uptake of D-glucose and 2-deoxy-D-glucose was greater in large cells from older animals than in small cells from young rats while at higher concentrations (3.0 mM–5.0 mM) uptake was similar. Insulin enhanced uptake of both sugars and the amounts accumulated by the two cell types were not significantly different. Also no difference was noted in basal rate of 3-O-methyl-D-glucose uptake or when uptake was accelerated by insulin stimulation. These findings suggest that large adipocytes from adult rats are not as insulin-resistant as previously suggested but, instead, have an efficient D-glucose transport system which is responsive to insulin stimulation.     

Use of tritiated 3-O-methyl-d-glucose for studies of membrane transport caveat

Tritiated 3-O-methyl-d-glucose has many useful attributes as a model substance for studies of the transport of glucose across cell membranes. However, preparations of high specific radioactivity can decompose within a few months, producing radioactive impurities that can cause a several-fold increase in the apparent rate of sugar transport. In our investigation radioactive contaminants entered frog skeletal muscle cells by free diffusion rather than by facilitated transport. Much of the contaminating radioactive material could be removed by evaporating the solvent and redissolving the sugar. Tritiated sugar samples that had a specific activity below 0.1 Ci/mmol remained stable and suitable for transport measurements after several years of storage at -20°C. In order to evaluate the suitability of a given tritiated preparation of sugar for transport measurements, it is recommended that its behavior be compared with that of a stable reference standard of low specific activity.     

Regulation of glucose transport by insulin and non-hormonal factors

Glucose uptake by nucleated cells is mediated by facilitated diffusion. In adipocytes, fibroblasts and muscle fibers uptake is regulated by a variety of hormones, environmental factors, and metabolic conditions. Glucose uptake by mammalian red cells also occurs by facilitated diffusion, but is not regulated by the same factors and conditions as in nucleated cells; yet the pharmacological and selectivity properties of this transport system resemble those of glucose uptake in regulated cells. The glucose transporter in the human red cell is a 55, 000 dalton protein, which has been purified to homogeneity and functionally reconstituted in artificial systems. Little is known about the molecular identity of the sugar carrier in other cell types. Glucose uptake is stimulated by insulin in muscle, fat and skin cells but not in bone, brain, placenta, erythrocytes nor probably lymphocytes. In responsive cells, stimulation occurs within seconds of exposure to the hormone; it requires cellular integrity but once elicited, it persists in isolated membranes; protein synthesis is not required for either the onset of the response or the return to basal conditions after hormone removal; on the other hand, intracellular energy is required for both steps; the cytoskeleton does not seem to be involved in the regulation of glucose uptake by insulin. In general, insulin increases V_t while K_t is unaffected. The hormone could affect the rate of turnover of the transporter in the membrane, and/or the number of transporters active at any time. An increase in the number of transport sites in the plasma membrane, due to incorporation of additional sites originating from intracellular membranes, has recently been proposed on the basis of both ³H-cytochalasin B binding and glucose transport determinations in isolated plasma and intracellular membranes. The feasibility and implications of a rapid and reversible translocation of glucose transport sites from specific intracellular pools to the plasma membrane are discussed.     

D-glucose permeability in river lamprey (Lampetra fluviatilis) and carp (Cyprinus carpio) erythrocytes.

1. The transport of 3-O-methyl-D-glucose (3-OMG) in lamprey and carp erythrocytes was studied. 2. In lamprey erythrocytes the half-time for the equilibration of 3-OMG was fast, approx. 8 min. In contrast, the erythrocytes of carp were almost impermeable to 3-OMG, with a half-time for equilibration of 14.2 hr. 3. 3-OMG was taken up by lamprey erythrocytes both by facilitated diffusion and simple diffusion. The presence of carrier-mediated transport was indicated by saturation kinetics and by inhibition by phloretin. The Km and Vm of the saturable component of 3-OMG transport were 1.6 mmol/l and 12.4 mmol/kg packed cells/hr. 4. Since the 3-OMG transport in carp erythrocytes showed no sign of saturation kinetics, it appears to proceed by simple diffusion only. 5. These results suggest that, as for most other teleost fish so far studied, low glucose permeability is insufficient to maintain normal energy metabolism in carp erythrocytes. In contrast, in agnathans facilitated glucose transport seems to be quite effective.     

Glucose transport and its inhibition by short-chain n-alkanes in Cladosporium resinae.

Glucose transport in Cladosporium resinae was studies with the aid of the non-metabolizable glucose analogue 3-O-methyl-D-glucose (3-O-MG). 3-O-MG, transported as a free sugar without phosphorylation, was found to inhibit glucose uptake competitively. Conversely, glucose was a competitive inhibitor of 3-O-MG uptake. Moreover, both glucose and 3-O-MG were able to bring about rapid counterflow intracellular 3-O-MG. Thus, glucose and 3-O-MG share the same entry and exit systems. The transport of 3-O-MG is carrier mediated and energy dependent as shown by saturation kinetics, strong temperature dependence, accumulation of unaltered 3-O-MG against a concentration gradient, and inhibition of uptake by NaN3, NaCN, and 2,4-dinitrophenol. The glucose transport system appeared to be constitutive for glucose transport in cells grown on fructose, galactose, mannose, xylose, or glucose. There was no derepressible low-Km glucose transport system in C. resinae. n-Hexane and n-heptane were found to inhibit 3-O-MG uptake rapidly at temperatures above 20 C. Over 50% inhibition of the uptake rate occurred after only 10 min of incubation with n-hexane at 30 C. The percentage of inhibition in the presence of n-hexane, compared to controls in the absence of n-hexane, was found to increase with increasing temperature. Longer-chain n-alkanes (C8 to C18) had no significant effect on uptake. The efflux of intracellular 3-O-MG, which appeared to occur by facilitated diffusion, was not affected by any of the n-alkanes tested including n-hexane.     

Isolation and properties of plasma membrane from smooth muscle

A generalized approach to obtain relatively pure fractions of plasma membrane from smooth muscle tissues for studying calcium transport is described. The use of various markers for cellular membranes to establish the purity of various fractions is critically considered. Plasma membranes from rat myometrium have been isolated in a purity estimated to be 95-99%. Plasma membrane purifications to 70-80% have been achieved from rat mesenteric arteries and veins, canine tracheal smooth muscle, rabbit intestinal muscle, rat vas deferens, rat fundus, and dog gastric corpus. The ATP-dependent transport of Ca is correlated with the distribution of plasma membrane markers. Ca gradient of greater than 1000-fold have been achieved. ATP-dependent active Ca transport by plasma membranes could sometimes be stimulated by oxalate or phosphate. Anion activation of Ca active transport is not a marker for endoplasmic reticulum. In some smooth muscles (e.g., rat vas deferens) ATP-dependent Ca uptake did not correlate exclusively with the distribution of plasma membrane markers. Instead, the correlation seemed to be with NADPH-cytochrome reductase EC 1.6.2.5 activity (putative endoplasmic reticulum marker) as well as with plasma membrane markers. In all smooth muscles, active Ca transport appears to be a property of the plasma membrane; in some it may also be a property of the endoplasmic reticulum. Mitochondria actively transport Ca, but in most systems studied to date, the Km for Ca2+ for this transport is higher than that for plasma membrane. Thus the plasma membrane may be the major physiological mechanism of active transport for Ca out of cytoplasm of smooth muscle cells. In two plasma membrane fractions (from rat myometrium and mesenteric arteries) it has been possible to demonstrate the existence of an Na-Ca exchange system. Its contribution to lowering cytoplasmic Ca is unknown.     

Reversal of insulin effects in fat cells may require energy for deactivation of glucose transport, but not deactivation of phosphodiesterase

The reversal of insulin effects on sugar transport and phosphodiesterase in fat cells was studied after arresting further actions of insulin with KCN, NaN₃, 2,4-dinitrophenol, or dicumarol. These agents rapidly lower the ATP concentration and concomitantly block the actions of insulin added later. Contrary to our expectation, the above inhibitors failed to initiate deactivation of the hormone-stimulated transport system. Instead, in the presence of the agents the transport system remained activated even after cells had been washed with an insulin-free buffer. This effect of the inhibitors was reversed when cells were washed with an inhibitor-free buffer containing glucose or pyruvate. The above inhibitors also blocked the deactivation of sugar transport stimulated by mechanical agitation. The effects of the inhibitors could not be explained by their possible effects on the basal transport activity, the intracellular urea space, or the cell count. The insulin-stimulated phosphodiesterase activity was rapidly lowered when cells were exposed to the above inhibitors. Apparently, these agents did not denature phosphodiesterase itself since the latter could be reactivated by insulin when inhibitor-treated cells were washed with a glucose-containing buffer. None of the above agents, except dicumarol, significantly inhibited phosphodiesterase activity in a cell-free system. It is suggested that the effects of insulin on sugar transport and phosphodiesterase are reversed by different mechanisms. ATP or metabolic energy may be involved in the deactivation of sugar transport, but not in that of phosphodiesterase.     

Regulation of the d-glucose transport system in isolated fat cells

Summary Recent technical advances have yielded considerable new biochemical insights into the hexose transport systems of both brown and white fat cells. In the present studies a novel filtration method was used to monitor initial rates of 3-O-(³H) methylglucose uptake in isolated white fat cells. Transport of 3-O-methylglucose, a non-metabolizable analogue of glucose, occurred by facilitated diffusion, was inhibited by glucose, phloridzin, cytochalasin B and dipyridamole, and was rapidly stimulated by insulin as well as lectins. Total 3-O-methylglucose uptake in white fat cells could be attributed to two kinetically distinct processes in addition to a certain degree of diffusion.Two important new features of glucose transport in fat cells have been discovered. First, in both brown and white fat cells transport per se does not appear to be necessarily rate-limiting for further glucose metabolism. Thus vitamin K₅, which markedly increases glucose oxidation by brown fat cells, did not affect the glucose transport system activity. Glucose utilization can apparently be significantly enhanced in fat cells by agents which either increase transport system activity or intracellular enzyme activity. Second, the transport system itself, whether in the basal state or after activation by insulin, lectins, or oxidants, is resistant to sulfhydryl reagents such as N-ethylmaleimide, while the increase in transport activity due to these agents is exquisitely sensitive to sulfhydryl blockage. N-ethylmaleimide blocks the stimulatory effect of insulin on transport whereas addition of insulin to fat cells prior to the reagent completely protects against this inhibitory effect. Further, N-ethylmaleimide prevents the elevated rates of transport system activity due to insulin (or other agents) from returning to basal levels once the cells are washed free of hormone. These data are consistent with the concept that activation of the transport system involves oxidation of key membrane sulfhydryls to the disulfide form, but alternative models are also possible. In any case, these findings provide a possible biochemical clue for future studies designed to identify the specific component(s) involved in the regulatory mechanism which modulates transport of glucose in isolated fat cells.Invited ArticleRecipient of the Elliot P. Joslin Research and Development Award of the American Diabetes Association.     

Counter-transport in chick embryo fibroblasts. A significant factor in measurement of glucose entry.

Enhanced rates of carrier-mediated 3-O-methyl-D-glucose (0.1 mM) transport were observed in primary cell cultures of chicken embryo fibroblasts deprived of glucose for 1 day. The addition of 5.5 mM-glucose, glucosamine or 2-deoxy-D-glucose for 15 min (37 degrees C) to glucose-starved cultures followed by washing and immediate measurement of 3-O-methyl-D-glucose transport resulted in an apparent further stimulation of transport. Transport stimulation increased with increasing concentrations of the added preincubation sugar and was observed at test concentrations ranging from 0.1 mM- to 10 mM-3-O-methyl-D-glucose. This enhancement occurred when the preloaded sugar was rapidly effluxing from cells and was eliminated by allowing cultures to incubate in buffer without sugar for 30 min (37 degrees C) after the removal of hexose and before measuring transport. A transient overshoot in the cumulative uptake of 3-O-methyl-D-glucose was observed in glucose-starved cultures that were pre-incubated in the presence of 55 mM-glucose or -glucosamine for 15 min (37 degrees C). These data suggest that counter-transport accounts for the apparent enhancement of glucose-transport capability observed in glucose-starved cells when they are briefly re-exposed to hexose.     

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.     

An Hg-sensitive channel mediates the diffusional component of glucose transport in olive cells

In several organisms solute transport is mediated by the simultaneous operation of saturable and non-saturable (diffusion-like) uptake, but often the nature of the diffusive component remains elusive. The present work investigates the nature of the diffusive glucose transport in Olea europaea cell cultures. In this system, glucose uptake is mediated by a glucose-repressible, H(+) -dependent active saturable transport system that is superimposed on a diffusional component. The latter represents the major mode of uptake when high external glucose concentrations are provided. In glucose-sufficient cells, initial velocities of D- and L-[U-(14)C]glucose uptake were equal and obeyed linear concentration dependence up to 100 mM sugar. In sugar starved cells, where glucose transport is mediated by the saturable system, countertransport of the sugar pairs 3-O-methyl-D-glucose/D-[U-(14)C]glucose and 3-O-methyl-D-glucose/3-O-methyl-D-[U-(14)C]glucose was demonstrated. This countertransport was completely absent in glucose-sufficient cells, indicating that linear glucose uptake is not mediated by a typical sugar permease. The endocytic inhibitors wortmannin-A and NH(4)Cl inhibited neither the linear component of D- and L-glucose uptake nor the absorption of the nonmetabolizable glucose analog 3-O-methyl-D-[U-(14)C]glucose, thus excluding the involvement of endocytic mediated glucose uptake. Furthermore, the formation of endocytic vesicles assessed with the marker FM1-43 proceeded at a very slow rate. Activation energies for glucose transport in glucose sufficient cells and plasma membrane vesicles were 7 and 4 kcal mol(-1), respectively, lower than the value estimated for diffusion of glucose through the lipid bilayer of phosphatidylethanolamine liposomes (12 kcal mol(-1)). Mercury chloride inhibited both the linear component of sugar uptake in sugar sufficient cells and plasma membrane vesicles, and the incorporation of the fluorescent glucose analog 2-NBDG, suggesting protein-mediated transport. Diffusive uptake of glucose was inhibited by a drop in cytosolic pH and stimulated by the protein kinase inhibitor staurosporine. The data demonstrate that the low-affinity, high-capacity, diffusional component of glucose uptake occurs through a channel-like structure whose transport capacity may be regulated by intracellular protonation and phosphorylation/dephosphorylation.     

Differential regulation of the GLUT-1 and GLUT-4 glucose transport systems by glucose and insulin in L6 muscle cells in culture

The regulation by glucose and insulin of the muscle-specific facilitative glucose transport system GLUT-4 was investigated in L6 muscle cells in culture. Hexose transport activity, mRNA expression, and the subcellular localization of the GLUT-4 protein were analyzed. As observed previously (Walker, P. S., Ramlal, T., Sarabia, V., Koivisto, U.-M., Bilan, P. J., Pessin, J. E., and Klip, A. (1990) J. Biol. Chem. 265, 1516-1523), 24 h of glucose starvation and 24 h of insulin treatment each increase glucose transport activity severalfold. Here we report a differential regulation of the GLUT-4 and GLUT-1 transport systems under these conditions. (a) The level of GLUT-4 mRNA was not affected by glucose starvation and was diminished by prolonged (24 h) administration of insulin; in contrast, the level of GLUT-1 mRNA was elevated under both conditions. (b) Glucose starvation and prolonged insulin administration increased the amount of both GLUT-4 and GLUT-1 proteins in the plasma membrane. (c) In intracellular membranes, glucose starvation elevated, and prolonged insulin administration reduced, the GLUT-4 protein content. In contrast, the GLUT-1 protein content in these membranes decreased with glucose starvation and increased with insulin treatment. Glucose transport was rapidly curbed upon refeeding glucose to glucose-starved cells, with half-maximal reversal after 30 min and maximal reversal after 4 h. This was followed by a marked decrease in the levels of GLUT-1 mRNA without major changes in GLUT-4 mRNA. Neither 2-deoxy-D-glucose nor 3-O-methyl-D-glucose could substitute for D-glucose in these effects. It is proposed that glucose and insulin differentially regulate the two glucose transport systems in L6 muscle cells and that the rapid down-regulation of hexose transport activity by glucose is regulated by post-translational mechanisms.