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.     

Regulation of hexose transport in BALB/c 3T3 preadipose cells: effects of glucose concentration and 12-O-tetradecanoylphorbol-13-acetate

Like many cell types in culture, both undifferentiated and differentiated BALB/c 3T3 preadipose cells respond to glucose deprivation with an increased uptake of 2-deoxy-D-glucose (deoxyglucose) and 3-O-methyl-D-glucose (methylglucose). Glucose readdition to glucose-deprived cultures resulted in a prompt fall in uptake activity; in undifferentiated cells, a half-maximally effective concentration of glucose was approximately 0.5 mM, while 0.1 mM was ineffective. Several hexoses differed in their efficacy of "deactivating" methylglucose transport in glucose-deprived cells; it appeared that a particular hexose must be metabolized beyond the 6-phosphate form to deactivate the transport system. Previous studies have shown that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulates hexose transport in undifferentiated and differentiated BALB/c 3T3 cells. In this study, it was found that TPA (and insulin in differentiated cells) prevented the glucose-induced deactivation of transport activity. Glucose-induced deactivation of transport activity was also prevented by cycloheximide or actinomycin D addition concomitantly with glucose. In glucose-starved cells, agents such as TPA and insulin appear to override a cellular control mechanism sensitive to the external concentration of glucose, so that elevated levels of transport activity are maintained under environmental conditions (i.e., a return to physiological glucose concentrations) that normally induce a fall in transport activity.     

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+.     

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.     

Expression of human glucose transporters in Xenopus oocytes: kinetic characterization and substrate specificities of the erythrocyte, liver, and brain isoforms

We describe the functional expression of three members of the family of human facilitative glucose transporters, the erythrocyte-type transporter (GLUT 1), the liver-type transporter (GLUT 2), and the brain-type transporter (GLUT 3), by microinjection of their corresponding mRNAs into Xenopus oocytes. Expression was determined by the appearance of transport activity, as measured by the transport of 3-O-methyl-D-glucose or 2-deoxy-D-glucose. We have measured the Km for 3-O-methyl-D-glucose of GLUTs 1, 2, and 3, and the results are discussed in light of the possible roles for these different transporters in the regulation of blood glucose. The substrate specificity of these transporter isoforms has also been examined. We show that, for all transporters, the transport of 2-deoxy-D-glucose is inhibited by D-but not by L-glucose. In addition, both D-galactose and D-mannose are transported by GLUTs 1-3 at significant rates; furthermore, GLUT 2 is capable of transporting D-fructose. The nature of the glucose binding sites of GLUTs 1-3 was investigated by using hexose inhibition of 2-deoxy-D-glucose uptake. We show that the characteristics of this inhibition are different for each transporter isoform.     

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.     

Highly insulin-responsive isolated rat heart muscle cells yielded by a modified isolation method

Freshly isolated adipocytes or cardiac myocytes appear to be subject to unspecific stimulation during isolation and subsequent handling, e.g. with respect to glucose transport. We have developed a modified procedure that yields rat cardiomyocytes with a very low basal, i.e. non stimulated hexose uptake rate (ca. 3 pmol * s-1 * mg protein-1 at 1 mM sugar), as compared to data reported by others. This low value correlates with the reported oxygen consumption of non-beating, isolated rat hearts, when these are perfused with glucose as the only substrate. The basal rate of glucose uptake in our quiescent cardiomyocytes is slightly lower than the value measured by others in beating rat hearts in vivo. Insulin (10 nM) stimulates 2-deoxy-D-glucose uptake 8- to 20-fold and 3-O-methyl-D-glucose uptake 14- to 20-fold, as compared to control. This insulin effect is markedly larger than that usually observed in isolated cardiomyocytes, but it is similar in magnitude to the stimulation of glucose transport reported for isolated, perfused rat hearts. In these cells, new stimulatory effects on the glucose transport, e.g. that of sulfhydryl reagents like phenylarsine oxide, become apparent. We conclude that the cardiomyocytes obtained by this modified method exhibit a basal glucose transport rate that is close to physiological values. These cells represent a new highly responsive model to detect and to investigate the effects of glucose transport stimulators (insulin, contraction etc.).     

Stimulation of hexose transport in L6 rat myoblasts by antibody and by glucose starvation.

Treatment of glucose-grown L6 rat myoblasts with rabbit or sheep anti-(L6-rat myoblast) antibody for 35 min or glucose starvation for at least 8 h results in a 2-fold increase in the Vmax. of 2-deoxy-D-glucose (dGlc) and 3-O-methyl-D-glucose uptake. In both cases, apparent transport affinities were not affected. Furthermore, once stimulation has occurred, further increases in hexose uptake could not be produced. Assays of antibody binding to whole cells suggested that the antibody is not internalized but remains bound on the cell surface. To elucidate the site and mechanism of antibody action, plasma-membrane vesicles from L6 cells were prepared. Anti-L6 antibody was found to cause a time- and dosage-dependent stimulation of dGlc transport in these vesicles. Maximum activation was achieved after 30 min exposure. This antibody-mediated activation could be inhibited by treatment of vesicles with various proteinase inhibitors. Treatment of vesicles with trypsin was also found to activate dGlc transport to levels observed with antibody. These results are virtually identical with those obtained with whole cells and suggest that antibody-mediated activation of hexose transport results from interaction of antibody with a specific membrane component(s).     

The coupling of metabolic to secretory events in pancreatic islets: does hexose transport affect cationic fluxes?

Poorly metabolized hexoses, such as 3-O-methyl-D-glucose, 2-deoxy-D-glucose and D-galactose failed to reproduce the inhibition of 86Rb outflow, the early inhibition and secondary rise in 45Ca efflux and the stimulation of insulin release evoked by D-glucose in perifused rat islets. Insulin release induced by either D-glucose or 2-ketoisocaproate was also unaffected by 3-O-methyl-D-glucose. It is concluded that hexose transport in islet cells does not represent in itself a significant determinant of the cationic and secretory response to D-glucose.     

Glucose metabolism in murine fetal cortical brain cells: lack of insulin effects

Glucose uptake and oxidation were markedly higher in cultured than in freshly isolated neural cells, prepared from murine fetal brain cortices. The hexose transport process--measured as 3-O-methyl-D-glucose uptake--appeared comparable in both conditions, and proceeded proportionally to the extracellular sugar concentration up to 6 mM. In contrast, glucose oxidation occurred independently of the prevailing glucose concentration from 1.4 mM on. Acute or chronic exposure to insulin exerted no effect upon cellular glucose uptake or oxidation. These results suggest that glucose handling by maturing fetal cortical cells is mainly determined by the rate of cellular glucose breakdown rather than by the rate of glucose transport into the cell; the marked rise in cellular glucose metabolism during culture might result from the synthesis and/or activation of a key enzyme in glucose catabolism. Our observations also indicate that the previously described neurotrophic effects of insulin are not mediated via enhanced glucose handling.     

Use of a genetic variant to study the hexose transport properties of human skin fibroblasts.

Human skin fibroblasts from 'normal' subjects were found to possess at least two hexose transport systems. One system was responsible for the uptake of 2-deoxy-D-glucose (dGlc), D-glucose and D-galactose, whereas the other was responsible primarily for the uptake of 3-O-methyl-D-glucose (MeGlc). The transport of dGlc was the rate-limiting step in the uptake process; over 97% of the internalized dGlc was phosphorylated and the specific activity of hexokinase was several times higher than that for dGlc transport. The dGlc transport system was activated by glucose starvation, and was very sensitive to inhibition by cytochalasin B and energy uncouplers. Fibroblasts isolated from a patient with symptoms of hypoglycaemia were found to differ from their normal counterparts in the dGlc transport system. They exhibited a much higher transport affinity for dGlc, D-glucose and D-galactose, with no change in the respective transport capacity. Transport was not the rate-limiting step in dGlc uptake by these cells. Moreover, the patient's dGlc transport system was no longer sensitive to inhibition by cytochalasin B and energy uncouplers. This suggested that the intrinsic properties of the patient's dGlc transport system were altered. It should be noted that the patient's dGlc transport system could still be activated by glucose starvation. Despite the changes in the dGlc transport system, the MeGlc transport system in the patient's fibroblasts remained unaltered. The observed difference in the properties of the two hexose transport systems in the 'normal' and the patient's fibroblasts strongly suggests that the two transport systems may be coded or regulated by different genes. The present finding provides the first genetic evidence from naturally occurring fibroblasts indicating the presence of two different hexose transport systems.     

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.     

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.     

Differences in glucose handling by pancreatic A- and B-cells

Glucose exerts opposite effects upon glucagon and insulin release from the endocrine pancreas. Glucose uptake and oxidation were therefore compared in purified A- and B-cells. In purified B-cells, the intracellular concentration of glucose or 3-O-methyl-D-glucose equilibrates within 2 min with the extracellular levels, and, like in intact islets, the rate of glucose oxidation displays a sigmoidal dose-response curve for glucose. In contrast, even after 5 min of incubation, the apparent distribution space of D-glucose or 3-O-methyl-D-glucose in A-cells remains much lower than the intracellular volume. In A-cells, both the rate of 3-O-methyl-D-glucose uptake and glucose oxidation proceed proportional to the hexose concentration up to 10 mM and reach saturation at higher concentrations. Addition of insulin failed to affect 3-O-methyl-D-glucose or D-glucose uptake and glucose oxidation by purified A-cells. Glucose releases 30-fold more insulin from islets than from single B-cells, but this marked difference is not associated with differences in glucose handling. The rate of glucose oxidation is virtually identical in single and reaggregated B-cells and is not altered after addition of glucagon or somatostatin. It is concluded that the dependency of glucose-induced insulin release upon the functional coordination between islet cells is not mediated through changes in glucose metabolism.     

The effect of triiodothyronine on insulin binding and action in rat adipocytes

The effects of a short term (2 hour) incubation of 5 microM triiodothyronine (T3) on 125I-insulin binding and insulin stimulated (14C)-2-deoxy-D-glucose uptake in rat adipocytes was investigated. In the presence of 5 microM T3, (14C)-2-deoxy-D-glucose uptake was significantly decreased by 11 to 24% at insulin concentrations of 5 to 1000 microU/ml. The concentration of insulin eliciting a half maximal response for insulin stimulated (14C)-2-deoxy-D-glucose uptake was 11.5 microU/ml in the control, and 14.3 microU/ml in the T3 treated cells (p less than 0.01). T3 treated adipocytes bound 9 to 22% less 125I-labeled insulin yet the concentration of native insulin necessary to displace 50% of the bound 125I-labeled insulin was the same in the control and T3 treated cells (75 and 70 ng/ml, respectively). These studies indicate that the decreased sensitivity of T3 treated cells to insulin is in accordance with a decreased number of receptors with the same binding characteristics as those of the control cells. The decreased maximal uptake of (14C)-2-deoxy-D-glucose at saturating insulin levels is likely to be independent of receptor number and result from a second, undetermined alteration in the hexose transport system of adipocytes treated with T3.     

Stimulation of glucose transport in osteoblastic cells by parathyroid hormone and insulin-like growth factor I

Insulin and parathyroid hormone (PTH) regulate glucose metabolism in bone cells. In order to differentiate between the effects of these hormones and to compare the potency of insulin with that of insulin-like growth factor (IGF) I, we treated rat bone-derived osteoblastic (PyMS) cells for different time periods and at different concentrations with insulin, IGF I, or PTH, and measured [1-(14)C]-2-deoxy-D-glucose (2DG) uptake and incorporation of D-[U-(14)C] glucose into glycogen. 2DG uptake was Na-independent with an apparent affinity constant (K (M)) of ~2 mmol/l. Expression of the high affinity glucose transporters (GLUT), GLUT1 and GLUT3 but not of GLUT4, was found by Northern and Western analysis. Similar to the findings with primary rat osteoblasts, but distinct from those in rat fibroblasts, 2DG uptake and glycogen synthesis were increased in this cell line after exposure to low concentrations (0.1 nmol/l and above) of PTH. IGF I at low doses (0.3 nmol/l and above) or insulin at higher doses (1 nmol/l and above) stimulated 2DG uptake and [(3)H] thymidine incorporation into DNA. 2DG transport was enhanced already after 30 min of IGF I treatment whereas the effect of PTH became significant after 6 h. It is concluded that IGF I rather than insulin may be a physiological regulator of 2DG transport and glycogen synthesis in osteoblasts.     

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

The membrane transport of glucose was studied in bovine adrenal chromaffin cell cultures by following the cell/medium distribution of the nonmetabolizable glucose analog, 3-O-methyl-D-glucose. Uptake of this sugar in day-1 cultures that are undergoing rapid morphological change and differentiation had a Vmax of 138 nmol/(mg protein.min) and Km of 15 mM, and was only slightly increased by 50 mU/mL insulin. In day-5 cultures where morphological changes were essentially completed, Vmax and Km decreased to 51 nmol/(mg protein.min) and 9.5 mM, respectively, and the response to insulin was restored to the level found in freshly isolated cells; this effect was abolished in the nominal absence of Ca2+. Thus, saturation kinetics and insulin and Ca2+ sensitivity of 3-methylglucose uptake observed in freshly isolated cells were maintained in culture. However, the insulin response was almost absent during the initial period of rapid morphological change when sugar transport was strongly stimulated. Culture of chromaffin cells in the presence of dexamethasone did not inhibit the formation of processes, but decreased 3-methylglucose uptake in day-5 cultures by an apparently competitive effect.     

Facilitated diffusion of monosaccharides in smooth muscle of rat vas deferens in vitro

The suitability of rat vas deferens for investigating sugar transport in smooth muscle was determined in vitro, with the nonmetabolized glucose analog 3-O-methyl-D-glucose as test sugar. Vas deferens smooth muscle contains a facilitated diffusion system for monosaccharides, as shown by saturation of the transport sites and by competition between 3-O-methyl-D-glucose and D-glucose. The activity of the facilitated diffusion system could be enhanced by hyperosmolarity and by contractile activity, but frequency dependency could not be established. A high concentration of insulin (100 mU/mL) was required to stimulate sugar transport. As smooth muscle is not a primary tissue for the storage of energy reserves, it does not require large numbers of insulin receptors.     

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.     

Stimulation of hexose transport in cultured human skin fibroblasts by insulin.

The effect of insulin on hexose transport in cultured human skin fibroblasts. Studies were carried out on cultures of human skin fibroblasts to explore the effect of insulin on hexose transport in serum-starved monolayers. Insulin (100 mU/ml) stimulated 2-deoxy-D-glucose transport (30% above control values) after 30 minutes exposure time, the response being similar up to four hours exposure to insulin. In several experiments (n = 22) employing three cell strains, insulin (100 mU/ml) exposure led to variable stimulation of 2-deoxy-D-glucose transport (an average of 37% above control values, with a range of 0 = 120%). The insulin-induced stimulation of 2-deoxy-D-glucose transport showed a dose dependency with increasing amounts of insulin, the response being maximal at an insulin concentration of 100 mU/ml. Kinetic analysis of 2-deoxy-D-glucose transport showed that insulin addition resulted in a slight change in the transport K_m (3.13 to 4.06 mM) and a 1.8-fold increase in the transport V_max (17.6 nanomoles/mg protein/min to 32.1 nanomoles/mg protein/min). Insulin also stimulated the transport of 3-0-methyl-D-glucose while the hexokinase activity of the cells was not affected. Further, this insulin-induced stimulation of sugar transport was not blocked by cycloheximide. The results indicate that insulin stimulated the stereospecific carrier-mediated of hexose transport in cultured human skin fibroblasts.