Increased glucose permeability in Babesia bovis-infected erythrocytes

Glucose influx into bovine erythrocytes was found to be significantly increased upon infection with the parasite, Babesia bovis. The influx of glucose into the infected cells over 4 min was not saturable at high concentrations of glucose (240 mM), nor was it affected by established inhibitors of mammalian glucose transport, such as cytochalasin B and phloretin (0.1-100 microM). Glucose uptake into the parasitized cells was, however, inhibited by phloridzin (phloretin-2-beta-glucoside) at concentrations over the range of 10-500 microM. Further inhibition of glucose uptake by adenosine (2.5-15 mM) was found to occur in B. bovis-infected bovine erythrocytes, suggesting an interaction of adenosine with the new or altered component of glucose transport in the parasitized cells.     

Effect of sphingoid bases on basal and insulin-stimulated 2-deoxyglucose transport in skeletal muscle.

Incubation of rat soleus muscles with 50 microM sphingosine or 50 microM sphinganine augmented basal 2-deoxy-D-glucose (2DG) transport 32%, but reduced the response to 0.1 and 1.0 mU insulin/ml by 17 and 27%, respectively. When the muscles were incubated with 50 microM phytosphingosine, a 63-93% increase in basal 2DG transport was observed. However, this treatment had no effect on insulin-stimulated 2DG transport. The phytosphingosine-induced increase in basal 2-DG transport was inhibited 93 and 98% with 35 and 70 microM cytochalasin B, respectively, suggesting that it is mediated by glucose transporters. Cellular accumulation of L-glucose, which is not mediated by glucose transporters, was not affected by phytosphingosine. It is concluded that (a) both sphingosine and sphinganine increase basal 2DG transport in muscle but diminish insulin-stimulated transport, and (b) phytosphingosine stimulates basal 2DG transport in muscle by a mechanism involving glucose transporters.     

Transport of 1,5-anhydro-D-glucitol into human polymorphonuclear leukocytes.

1,5-Anhydro-D-glucitol (AG) is one of the main polyols and its structure resembles glucose. It has been proposed that decreased serum AG concentrations in diabetic patients are a novel indicator of diabetic metabolic derangement. However, the pathway of AG metabolism still remains to be clarified. In this study we investigated the transport of AG into human polymorphonuclear leukocytes (PMNLs) isolated from healthy volunteers and found that 0.1 mM 3-O-methy-D-glucose (3OMG) was equilibrated with a half saturation time of 10 s, while the uptake rate of AG was much slower. The concentration dependence of AG uptake revealed that the AG transport velocity reached a plateau, with a Km of about 50 mM and Vmax of about 25 nmol/min/10(7) cells. Transport of 14C-labeled 3OMG was inhibited by unlabeled D-glucose or AG in a dose-dependent manner. The mean inhibition constant (Ki) for D-glucose and for AG were 1.06 and 4.93 mM, respectively. Cytochalasin B (20 microM) inhibited 3OMG transport by 90% but AG transport by only 50%. S/V for 14C-labeled AG transport plotted against the concentration of unlabeled 3OMG showed a non-linear and biphasic pattern. These results suggest that AG influx into PMNLs is mediated not only by the cytochalasin B-sensitive glucose transport system but also via another facilitated transport system.     

Acute effects of troglitazone and nitric oxide on glucose uptake in L929 fibroblast cells

The thiazolidinedione class of antidiabetic drugs, including troglitazone, has an insulin-sensitizing effect for patients with type 2 diabetes. However, in some tissues, studies have shown that troglitazone also has an acute insulin-independent effect on glucose uptake. To determine the extent of this acute action of troglitazone, the effect of troglitazone on 2-deoxyglucose (2DG) uptake in L929 fibroblast cells was measured. Troglitazone stimulated 2DG uptake in a dose dependent manner with a maximum stimulation of >300% at 5-10 microM. In addition, nitric oxide has been shown to stimulate glucose uptake in peripheral muscle tissue. Therefore, the effect of nitric oxide on 2DG uptake in L929 cells was also investigated using the nitric oxide donor, sodium nitroprusside (SNP). SNP stimulated 2DG uptake by >200% with a maximally effective concentration of 5 mM. The combined effect of maximally effective concentrations of both stimulants (10 microM troglitazone + 5 mM SNP) was not additive suggesting a shared pathway for 2DG uptake. However, the nitric oxide synthase inhibitor, N(G)-monomethyl-L-arginine (L-NMMA, 50 microM) had no effect on troglitazone stimulated 2DG uptake, indicating that the troglitazone and nitric oxide pathways converge after nitric oxide production. In addition, 12.5 microM dantrolene was shown to have no effect on either troglitazone or SNP stimulated 2DG uptake suggesting that these stimulatory effects are independent of changes in calcium ion concentrations. These data provide important evidence for the acute regulation of glucose transport through GLUT 1 transporters.     

Uptake and metabolism of d‐glucose in isolated acinar and ductal cells from rat submandibular glands

The present study deals with the possible effects of selected environmental agents upon the uptake and metabolism of d ‐glucose in isolated acinar and ductal cells from the rat submandibular salivary gland. In acinar cells, the uptake of d ‐[U‐¹⁴C]glucose and its non‐metabolised analogue 3‐O‐[¹⁴C‐methyl]‐d ‐glucose was not affected significantly by phloridzin (0.1 mM) or substitution of extracellular NaCl (115 mM) by an equimolar amount of CsCl, whilst cytochalasin B (20 μM) decreased significantly such an uptake. In ductal cells, both phloridzin and cytochalasin B decreased the uptake of d ‐glucose and 3‐O‐methyl‐d ‐glucose. Although the intracellular space was comparable in acinar and ductal cells, the catabolism of d ‐glucose (2.8 or 8.3 mM) was two to four times higher in ductal cells than in acinar cells. Phloridzin (0.1 mM), ouabain (1.0 mM) and cytochalasin B (20 μM) all impaired d ‐glucose catabolism in ductal cells. Such was also the case in ductal cells incubated in the absence of extracellular Ca²⁺ or in media in which NaCl was substituted by CsCl. It is proposed that the ductal cells in the rat submandibular gland are equipped with several systems mediating the insulin‐sensitive, cytochalasin B‐sensitive and phloridzin‐sensitive transport of d ‐glucose across the plasma membrane. Copyright © 2014 John Wiley & Sons, Ltd.     

Regulation of hexose transport in L8 myocytes by glucose: possible sites of interaction

Previous work demonstrated that glucose controls its own transport rate in rat skeletal muscle: exposure to high glucose levels down-regulates muscle hexose transport, while glucose withdrawal results in elevated transport rates (J. Biol. Chem. 261:16827-16833, 1986). The present study investigates the mechanism of this autoregulatory system. Preincubation of L8 myocytes at 16 mM glucose reduced subsequent 2-deoxy-D-glucose (dGlc) uptake by 40% within 3 h. Cycloheximide (1 microM) mimicked the action of glucose; the effects of glucose and cycloheximide were not additive. At 50 microM, cycloheximide prevented the modulations of glucose transport induced by exposure of muscle cells to high or low glucose concentrations. Inhibition of glycosylation with tunicamycin A1 reduced the basal dGlc uptake, but did not prevent its up-regulation following glucose withdrawal. Inhibition of RNA synthesis by actinomycin D prevented the down-regulatory effect of glucose. These results indicate that continuous protein synthesis and protein glycosylation are required for the maintenance of the steady-state dGlc uptake. We suggest that glucose exerts its autoregulatory effect on hexose transport by modifying the incorporation of active glucose transporters into the plasma membrane rather than changing their rate of degradation. It is hypothesized that this effect is mediated by a non-glycosylated protein involved in the translocation or activation of glucose transporters.     

Uptake of glycerol by tumor cells and its control by glucose.

Glycerol can be utilized by cultured cells of Novikoff rat hepatoma, Hela and HEP-2. Glucose inhibits the rate of glycerol uptake by the first two cell lines but stimulates the process in the third. The transport process of glycerol, particularly by its insensitivity to phloridzin, is distinguishable from that of glucose. The inhibitory effect of glucose on glycerol uptake which is competitive in nature is at the membrane transport and not phosphorylation step, since $\text{in vitro}$ glycerol kinase is sensitive to neither glucose nor hexose phosphates.     

Calcium-independent inhibition of glucose transport in PC-12 and L6 cells by calcium channel antagonists

The goal of these studies was todetermine whether different calcium channel antagonists affect glucosetransport in a neuronal cell line. Rat pheochromocytoma (PC-12) cellswere treated with L-, T-, and N-type calcium channel antagonists beforemeasurement of accumulation of 2-[³H]deoxyglucose(2-[³H]DG). The L-type channel antagonistsnimodipine, nifedipine, verapamil, and diltiazem all inhibited glucosetransport in a dose-dependent manner (2-150 µM) withnimodipine being the most potent and diltiazem only moderatelyinhibiting transport. T- and N-type channel antagonists had no effecton transport. The L-type channel agonist l-BAY K 8644 alsoinhibited uptake of 2-[³H]DG. The ability of these drugsto inhibit glucose transport was significantly diminished by thepresence of unlabeled 2-DG in the uptake medium. Some experiments wereperformed in the presence of EDTA (4 mM) or in uptake buffer withoutcalcium. The absence of calcium in the uptake medium had no effect oninhibition of glucose transport by nimodipine or verapamil. To examinethe effects of these drugs on a cell model of a peripheral tissue, westudied rat L6 muscle cells. The drugs inhibited glucose transport in L6 myoblasts in a dose-dependent manner that was independent of calciumin the uptake medium. These studies suggest that the calcium channelantagonists inhibit glucose transport in cells through mechanisms otherthan the antagonism of calcium channels, perhaps by acting directly onglucose transporters.

     

Effect of insulin on the glucose utilization in isolated cardiac myocytes from adult rat

The acute effects of insulin on glucose utilization in isolated rat quiescent cardiac myocytes were studied. Insulin (80 nM) increased the rate of glucose clearance by 2-3 times in the presence of glucose ranging from 0.3 microM to 5.5 mM. Glucose transport, which was measured in terms of both D-glucose uptake in the presence of 0.3 microM D-glucose and initial rate of uptake of 3-O-methylglucose, was stimulated 3-fold in the presence of insulin. At higher glucose concentrations (greater than 100 microM), a decrease in glucose clearance rate due to a shift of the rate-limiting step from glucose transport to a post-transport step in the pathway of glucose metabolism was observed. At the physiological concentration of glucose (5.5 mM), about 73% of glucose was metabolized into lactate, about 10% was oxidized into CO2 and the rest (17%) remained inside the cells. The pentose phosphate pathway did not contribute to the glucose metabolism in these cells. Insulin (80 nM) significantly increased the uptake of glucose (112%), and the conversions of glucose into lactate (16%), glycogen (64%), and triglyceride (18%), but not into CO2 (3%). Insulin transiently increased the percentage of I-form of glycogen synthase by 16% above basal, but did not affect the percentage of a-form of glycogen phosphorylase. The content of glucose 6-phosphate in the cells was increased by 46% above the basal value in the presence of insulin. These results indicate that insulin has different acute stimulatory effects on various steps in the metabolic pathway of glucose in isolated quiescent cardiac myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monosaccharide uptake in common carp (Cyprinus carpio) EPC cells is mediated by a facilitative glucose carrier

In most animal cells, transport of monosaccharides across the plasma membrane is mediated by glucose transporters (GLUT). Mammals express at least five distinct transporters (GLUTs 1--5), which are well characterised both functionally and genetically. In contrast, the glucose transport system of fish remains poorly studied. Here we report studies of hexose uptake in carp EPC cells and cloning of a glucose transporter cDNA from these cells. Transport of radio-labelled methylglucose (3-OMG) followed Michaelis--Menten kinetics with a K(m) value (8.5 mM) similar to that of mammalian cells. The inhibition of transport by cytochalasin B and phloretin, but not by phloridzin or cyanide, strongly suggested the existence of a facilitative carrier. D-Glucose, 2-deoxyglucose, 3-OMG, D-mannose and D-xylose were competitive inhibitors of 3-OMG uptake, while L-glucose, mannitol, D-fructose, D-ribose and sucrose did not compete with 3-OMG. We cloned a carp glucose transporter (CyiGLUT1), using RT-PCR and RACE strategies. CyiGLUT1 was different from known carp and zebrafish EST sequences. The complete cDNA (3060 bp) contained one open reading frame encoding a predicted protein of 478 amino acids. The deduced amino acid sequence shared 78% identity with mammalian and avian GLUT1 proteins. Key amino acids involved in substrate selection and catalysis of mammalian GLUTs were conserved in the carp transporter.     

Transport of 1,5-anhydro-D-glucitol into insulinoma cells by a glucose-sensitive transport system

The uptake of 1,5-anhydro-D-glucitol (1,5-AG) occurs by passive mechanisms in cells or tissues that have passive glucose transporters. It is known that serum 1,5-AG concentrations are reduced in patients with diabetes mellitus. To elucidate the metabolism of this substance and its physiological role in pancreatic β-cells, we assayed 1,5-AG transport in the insulinoma-derived cell lines, RINr and MIN6. Both cell lines showed an insulin-insensitive, concentration-dependent uptake of 1,5-AG with a saturation time of approximately 120 min, and most of the 1,5-AG in the cytoplasm was in the free form. A biphasic saturation curve was obtained using a wide range of 1,5-AG concentrations, suggesting that accumulation was mediated by a high affinity and a low affinity transporter. The high affinity transporter had a K_m of 10.4 in RINr cells and 13.0 mM in MIN6 cells, and the low affinity transporter had a K_m of 131 in RINr cells and 211 mM in MIN6 cells. Uptake of 1,5-AG was markedly inhibited by phloretin and cytochalasin B, but was only slightly affected by phloridzin. Uptake of 1,5-AG was markedly inhibited by glucose at physiological concentrations (1.0–10 mM), as well as by galactose and mannose. The 1,5-AG concentration required to inhibit 2-deoxyglucose uptake exceeded that of glucose by >100 times, being much higher than the physiological concentrations of 1,5-AG. These results indicate that the 1,5-AG carrier system in insulinoma cells is distinct from that in either the somatic cells or renal tubular cells. These findings also suggest that a unique 1,5-AG transport system is present in pancreatic β-cells.     

Some properties of the thiamine uptake system in isolated rat hepatocytes

A kinetic study of [14C]thiamine uptake over a concentration range from 0.1 microM to 4 mM was performed in isolated rat hepatocytes. The results showed that two processes contribute to the entry in rat hepatocytes: a low affinity process with a Kt of 34.1 microM and Vmax of 20.8 pmol/10(5) cells per 30 s and a high affinity process with a Kt of 1.26 microM and Vmax of 1.21 pmol/10(5) cells per 30 s. The uptake of thiamine by the high affinity process was concentrative and reduced in a betaine medium or K+ medium. Both ouabain and 2,4-dinitrophenol decreased the thiamine uptake by the high affinity process. These findings indicate that the transport of thiamine via a high affinity process is dependent on Na+ and biological energy. The uptake of thiamine was strongly inhibited by thiamine analogs such as dimethialium and chloroethylthiamine. Among quarternary ammonium compounds other than thiamine derivatives, choline and acetylcholine significantly inhibited thiamine uptake by rat liver cells, whereas betaine and carnitine did not. A kinetic study of thiamine uptake by rat hepatocytes preloaded with pyrithiamine, a potent inhibitor of thiamine pyrophosphokinase, revealed that the biphasic property of thiamine uptake disappeared and a single carrier system for thiamine with a Kt of 40.5 microM, which was similar to the Kt value of the low affinity process, was retained. These results strongly suggest that thiamine transport system in rat liver cells is closely connected with thiamine pyrophosphokinase, which accelerates the uptake rat of thiamine by pyrophosphorylation at physiological concentrations of thiamine.     

Characteristics of Glucose Transport in Neuronal Cells and Astrocytes from Rat Brain in Primary Culture

Glucose transport systems in cultured neuronal cells and astrocytes of rats were characterized by measuring the uptake of 2-deoxy-D-[3H]glucose ([3H]2-DG) into the cells. Various sugars inhibited 2-DG uptake by neuronal cells and astrocytes similarly, a finding indicating that the substrate specificities of the transporters in the two types of cells were almost the same. However, the Km values for 2-DG of neuronal cells and astrocytes were 1.7 and 0.36 mM, respectively. The uptake of 2-DG was strongly inhibited by cytochalasin B. Nucleosides, such as adenosine, inosine, and uridine, inhibited 2-DG uptake competitively in both neuronal cells and astrocytes. The uptake by both types of cells were also inhibited by forskolin, but not by cyclic AMP, an observation suggesting that forskolin bound directly to the transporters to cause inhibition. Its inhibition was competitive in astrocytes and noncompetitive in neuronal cells. Astrocytes contained a glucose transporter with a subunit molecular weight of 45K, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after photoaffinity labeling using [3H]cytochalasin B as a probe.     

Methylene blue stimulates 2-deoxyglucose uptake in L929 fibroblast cells

Methylene blue (MB), a common cell stain, has been shown to inhibit nitric oxide synthase and guanylate cyclase, which has led to the recent use of MB in nitric oxide signaling studies. This study documents the effects of MB on 2-deoxyglucose (2DG) uptake in L929 fibroblast cells where uptake is controlled by a single glucose transporter, GLUT 1. MB significantly activates cytochalasin B-inhibitable glucose transport in a dose dependent fashion within 10 min. A maximal stimulation of up to 800% was achieved by 50 microM MB after a 45-min exposure. The Vmax of transport increased without a change in the Km, which was accomplished without a significant change in the GLUT 1 content. The reduced form of MB, did not stimulate 2DG uptake and potassium ferricyanide, an extracellular redox agent, prevented both the staining and stimulatory effects of MB suggesting MB is reduced at the cell surface before it enters L929 cells. Phenylarsine oxide did not block cell staining as noted in other cells lines, but it did inhibit both basal and MB-stimulated 2DG uptake. Likewise, methyl-beta-cyclodextrin, an agent used to remove membrane cholesterol, blocked both the staining and stimulatory effects of MB. The AMP analog, AICAR, inhibited rather than activated basal 2DG uptake, and it did not alter MB-stimulated uptake suggesting that AMP kinase activation is not critical to the MB effect. Wortmannin, an inhibitor of PI kinase, had no effect on MB-stimulated 2DG uptake. These data provide additional insight into the acute regulation of GLUT 1 transport activity in L929 cells.     

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.     

Alternative pathways of glucose transport in Prevotella bryantii B(1)4(1)

Prevotella bryantii B(1)4 grew faster on glucose than mannose (0.70 versus 0.45 h(-1)), but these sugars were used simultaneously rather than diauxically. 2-deoxy-glucose (2DG) decreased the growth rate of cells that were provided with either glucose or mannose, but 2DG did not completely prevent growth. Cells grown on glucose or mannose transported both (14)C-glucose and (14)C-mannose, but cells grown on glucose had over three-fold higher rates of (14)C-glucose transport than cells grown on mannose. The (14)C-mannose transport rates of glucose- and mannose-grown cells were similar. Woolf-Augustinsson-Hofstee plots were not linear, and it appeared that the glucose/mannose/2DG carrier acted as a facilitated diffusion system at high substrate concentrations. When cultures were grown on nitrogen-deficient (excess sugar) medium, isolates had three-fold lower (14)C-glucose transport, but the (14)C-mannose transport did not change significantly. (14)C-glucose and (14)C-mannose transport rates could be inhibited by 2DG and either mannose or glucose, respectively. The (14)C-glucose transport of mannose-grown cells was inhibited more strongly by mannose and 2DG than those grown on glucose. Cells grown on glucose or mannose had similar ATP-dependent glucokinase activity, and 2DG was a competitive inhibitor (K(i)=0.75 mM). Thin layer chromatography indicated that cell extracts also had ATP-dependent mannose phosphorylation, but only a small amount of phosphorylated 2DG was detected. Glucose, mannose or 2DG were not phosphorylated in the presence of PEP. Based on these results, it appeared that P. bryantii B(1)4 had: (1) two mechanisms of glucose transport, a constitutive glucose/mannose/2DG carrier and an alternative glucose carrier that was regulated by glucose availability, (2) an ATP-dependent glucokinase that was competitively inhibited by 2DG but was unable to phosphorylate 2DG at a rapid rate, and (3) virtually no PEP-dependent glucose, mannose or 2DG phosphorylation activities.     

Na+-linked active transport of ascorbate into cultured bovine retinal pigment epithelial cells: heterologous inhibition by glucose

The transport of ascorbate into cultured bovine retinal pigment epithelial (RPE) cells is reported. Primary or subcultured RPE cells were incubated in the presence of 10-500 microM L-[carboxyl-14C]-ascorbate for various periods of time. Accumulation of ascorbate into RPE cells followed a saturable active transport with a Km of 125 microM and a Vmax of 28 pmole/micrograms DNA/min. RPE intracellular water was calculated to be 0.8 pL/cell, and the transported cellular ascorbate concentration was 7.5 +/- 0.8 mM. Replacement of 150 mM NaCl in the incubation media with choline-Cl strongly inhibited (80 +/- 8%) ascorbate uptake into cultured RPE cells. Although the depletion of cellular ATP by 2,4-dinitrophenol and the inhibition of Na+-K+-ATPase by ouabain reduced ascorbate transport into RPE significantly, active transport of ascorbate was not entirely inhibited by these metabolic inhibitors. The ascorbate analogue, D-isoascorbate, competitively inhibited ascorbate transport into cultured RPE with a Ki of 12.5 mM. Cells grown in the presence of 5 to 50 mM alpha-D-glucose in the growth media did not differ in their ability to transport ascorbate. In contrast, the presence of alpha-D-glucose or its nonmetabolizable analogues, 3-0-methyl-glucose, alpha-methyl-glucose, and 2-deoxy-glucose, but not L-glucose or beta-D-fructose, in the incubation media inhibited ascorbate transport. myo-Inositol (10 or 20 mM) also inhibited ascorbate transport into RPE cells. The active uptake of ascorbate into cultured RPE cells was primarily coupled to the movement of sodium ion down its electrochemical gradient. A bifunctional, cotransport carrier possessing an ascorbate-binding site and a sodium-binding site may be involved in the ascorbate uptake system. The inhibition of ascorbate uptake by sugars appeared to be heterologous in nature, occurring between two distinct carrier systems, both of which were dependent on the sodium ions.     

L-Fucose Is a Potent Inhibitor of myo-Inositol Transport and Metabolism in Cultured Neuroblastoma Cells

It has been proposed that abnormal myo-inositol metabolism may be a factor in the development of diabetic complications. Studies with animal models of diabetes and cultured cells have suggested that hyperglycemia by an unknown mechanism may alter myo-inositol metabolism and content. Recently, we have shown that L-fucose, a 6-deoxy sugar whose content has been reported to be increased in diabetes, is a potent inhibitor of myo-inositol transport. To examine the effect of L-fucose on myo-inositol metabolism, neuroblastoma cells were cultured in medium supplemented with L-fucose. L-Fucose is a competitive inhibitor of Na(+)-dependent, high-affinity myo-inositol transport. The Ki for inhibition of myo-inositol transport by L-fucose is about 3 mM. L-Fucose is taken up and accumulates in neuroblastoma cells. The uptake of L-fucose is inhibited by Na+ depletion, D-glucose, glucose analogues, phloridzin, and cytochalasin B. In contrast, neither myo-inositol nor L-glucose inhibits L-fucose uptake. Chronic exposure of neuroblastoma cells to 1-30 mM L-fucose causes a decrease in myo-inositol accumulation and incorporation into inositol phospholipids, intracellular free myo-inositol content, and phosphatidylinositol levels. Na+,K(+)-ATPase transport activity is decreased by about 15% by acute or chronic exposure of neuroblastoma cells to L-fucose. Similar defects occur when neuroblastoma cells are exposed chronically to 30 mM glucose. Cell myo-inositol metabolism and Na+/K(+)-pump activity are maintained when 250 microM myo-inositol is added to the L-fucose-supplemented medium. Unlike the effect of chronic exposure of neuroblastoma cells to medium containing 30 mM glucose, the resting membrane potential of neuroblastoma cells is not altered by chronic exposure of the cells to 30 mM L-fucose. The effect of L-fucose on cultured neuroblastoma cell properties occurs at concentrations of L-fucose which may exist in the diabetic milieu. These data suggest that increased concentrations of L-fucose may have a role in myo-inositol-related defects in mammalian cells.     

myo-Inositol Transport in Mouse Astroglia-Rich Primary Cultures

Uptake of radiolabeled myo-inositol was studied in astroglia-rich primary cultures derived from neonatal mouse brains. The uptake was saturable in the presence of Na+ with a Km of 25 microM and a Vmax of 60 pmol.min-1.(mg protein)-1, suggesting a high-affinity transport system for myo-inositol in astroglial cells. In addition, a Na(+)-independent, nonsaturable component was found. Carrier-mediated uptake was not inhibited by cytochalasin B (50 microM), but was reduced by depolarizing concentrations of K+ and, to different extents, in the presence of phloretin, ouabain, or amiloride (1 mM each). scyllo-Inositol, glucose, and galactose also reduced myo-inositol uptake; inhibition by the two hexoses was not reversed in the presence of 0.4 mM sorbinil. On the other hand, uptake of 2-deoxyglucose was not inhibited by high concentrations of myo-inositol. Preincubation of the cells with glucose-free or inositol-free medium stimulated uptake of myo-inositol and preincubation with 25 mM glucose in the presence of 0.4 mM sorbinil had no effect on the rate of uptake. The results suggest that myo-inositol is taken up into the astroglial cells by a transport mechanism that is distinct from that of glucose and probably is an active one. Sorbitol pathway activity does not interfere with myo-inositol uptake.     

Inhibition of insulin-stimulated glucose transport in rat adipocytes by nucleoside transport inhibitors

In isolated rat adipocytes, basal as well as insulin-stimulated 3-O-methylglucose transport was inhibited nearly completely (maximal inhibition: 95%) by the nucleoside transport inhibitors dipyridamole (IC50 = 5 microM), nitrobenzylthioguanosine (20 microM), nitrobenzylthioinosine (35 microM) and papaverine (130 microM). Transport kinetics in the presence of 10 microM dipyridamole revealed a significant increase in the transport Km value of 3-O-methylglucose (3.45 +/- 0.6 vs 2.36 +/- 0.29 mM in the controls) as well as a decrease in the Vmax value (4.84 +/- 0.95 vs 9.03 +/- 1.19 pmol/s per microliter lipid in the controls). Half-maximally inhibiting concentrations of dipyridamole were one order of magnitude higher than those inhibiting nucleoside (thymidine) uptake (0.48 microM). The inhibitory effect of dipyridamole (5 microM) reached its maximum within 30 s. The agent failed to affect insulin's half-maximally stimulating concentration (0.075 nM) indicating that it did not interfere with the mechanism by which insulin stimulates glucose transport. Further, dipyridamole fully suppressed the glucose-inhibitable cytochalasin B binding (IC50 = 1.65 +/- 0.05 microM). The data indicate that nucleoside transport inhibitors reduce glucose transport by a direct interaction with the transporter or a closely related protein. It is suggested that glucose and nucleoside transporters share structural, and possibly functional, features.