Cellular binding and uptake of fluorescent glucose analogs 2-NBDG and 6-NBDG occurs independent of membrane glucose transporters.

The classical methods for determining glucose uptake rates in living cells involve the use of isotopically labeled 2-deoxy-d-glucose or 3-O-methyl-d-glucose, which enter cells via well-characterized membrane transporters of the SLC2A and SLC5A families, respectively. These classical methods, however, are increasingly being displaced by high-throughput assays that utilize fluorescent analogs of glucose. Among the most commonly used of these analogs are 2-NBDG and 6-NBDG, which contain a bulky 7-nitro-2,1,3-benzoxadiazol-4-yl-amino moiety in place of a hydroxy group on d-glucose. This fluorescent group significantly alters both the size and shape of these molecules compared to glucose, calling into question whether they actually enter cells by the same transport mechanisms. In this study, we took advantage of the well-defined glucose uptake mechanism of L929 murine fibroblasts, which rely exclusively on the Glut1/Slc2a1 membrane transporter. We demonstrate that neither pharmacologic inhibition of Glut1 nor genetic manipulation of its expression has a significant impact on the binding or uptake of 2-NBDG or 6-NBDG by L929 cells, though both approaches significantly impact [3H]-2-deoxyglucose uptake rates. Together these data indicate that 2-NBDG and 6-NBDG can bind and enter mammalian cells by transporter-independent mechanisms, which calls into question their utility as an accurate proxy for glucose transport.

NMS-873 functions as a dual inhibitor of mitochondrial oxidative phosphorylation.

Small-molecule inhibitors of enzyme function are critical tools for the study of cell biological processes and for treatment of human disease. Identifying inhibitors with suitable specificity and selectivity for single enzymes, however, remains a challenge. In this study we describe our serendipitous discovery that NMS-873, a compound that was previously identified as a highly selective allosteric inhibitor of the ATPase valosin-containing protein (VCP/p97), rapidly induces aerobic fermentation in cultured human and mouse cells. Our further investigation uncovered an unexpected off-target effect of NMS-873 on mitochondrial oxidative phosphorylation, specifically as a dual inhibitor of Complex I and ATP synthase. This work points to the need for caution regarding the interpretation of cell survival data associated with NMS-873 treatment and indicates that cellular toxicity associated with its use may be caused by both VCP/p97-dependent and VCP/p97-independent mechanisms.

Pharmacologic inhibition of N-linked glycan trimming with kifunensine disrupts GLUT1 trafficking and glucose uptake.

The facilitative glucose transport GLUT1 (SLC2A1) is a constitutively expressed membrane protein involved in basal uptake of blood glucose. GLUT1 modification by N-linked glycosylation at a single asparagine residue (N45) appears to play multiple roles in the trafficking, stability and transport activity of this protein. Here we examine the role of complex N-glycosylation on GLUT1 function in renal epithelial cells by arresting this modification at the high-mannose stage with the mannosidase I inhibitor kifunensine. Consistent with prior work in which GLUT1 glycosylation was completely inhibited, we find that kifunensine treatment results in a time-dependent decrease of up to 40% in cellular glucose uptake. We further demonstrate that this effect is primarily a result of deficient GLUT1 trafficking to the cell membrane due to quality control mechanisms that instead direct GLUT1 to the ER-associated degradation (ERAD) pathway. Unlike tunicamycin, which inhibits the first step in N-glycosyl transfer and causes dramatic cell cycle arrest, kifunensine causes only a modest decrease in GLUT1 levels and cell cycle progression in both normal and transformed renal cells. The effect of kifunensine on the cell cycle appears to be independent of its effect on GLUT1, since all renal cell types in this study displayed decreased proliferation regardless of their dependence on glucose uptake for growth and survival. Together these results indicate that proper N-glycan processing plays an important role in directing GLUT1 to the cell surface and that disruption of mannosidase activity results in aberrant degradation of GLUT1 by the ERAD pathway.

GLUT1 is associated with sphingolipid-organized, cholesterol-independent domains in L929 mouse fibroblast cells.

Glucose is a preferred metabolite in most mammalian cells, and proper regulation of uptake is critical for organism homeostasis. The glucose transporter 1 (GLUT1) is responsible for glucose uptake in a wide variety of cells and appears to be regulated in a tissue specific manner. Therefore, a better understanding of GLUT1 regulation within its various cellular environments is essential for developing therapeutic strategies to treat disorders associated with glucose homeostasis. Previous findings suggest that plasma membrane subdomains called lipid rafts may play a role in regulation of GLUT1 uptake activity. While studying this phenomenon in L929 mouse fibroblast cells, we observed that GLUT1 associates with a low density lipid microdomain distinct from traditionally-defined lipid rafts. These structures are not altered by cholesterol removal with methyl-ß-cyclodextrin and lack resistance to cold Triton X-100 extraction. Our data indicate that the GLUT1-containing membrane microdomains in L929 cells, as well as GLUT1's basal activity, are instead sphingolipid-dependent, being sensitive to both myriocin and sphingomyelinase treatment. These microdomains appear to be organized primarily by their lipid composition, as disruption of the actin cytoskeleton or microtubules does not alter the association of GLUT1 with them. Furthermore, the association of GLUT1 with these microdomains appears not to require palmitoylation or glycosylation, as pharmacologic inhibition of these processes had no impact on GLUT1 density in membrane fractions. Importantly, we find no evidence that GLUT1 is actively translocated into or out of low density membrane fractions in response to acute activation in L929 cell.

Quercetin inhibits glucose transport by binding to an exofacial site on GLUT1.

Quercetin, a common dietary flavone, is a competitive inhibitor of glucose uptake and is also thought to be transported into cells by GLUT1. In this study, we confirm that quercetin is a competitive inhibitor of GLUT1 and also demonstrate that newly synthesized compounds, WZB-117 and BAY-876 are robust inhibitors of GLUT1 in L929 cells. To measure quercetin interaction with L929 cells, we develop a new fluorescent assay using flow cytometry. The binding of quercetin and its inhibitory effects on 2-deoxyglucose (2DG) uptake showed nearly identical dose dependent effects, with both having maximum effects between 50 and 100 µM and similar half maximum effects at 8.9 and 8.5 µM respectively. The interaction of quercetin was rapid with t1/2 of 54 s and the onset and loss of its inhibitory effects on 2DG uptake were equally fast. This suggests that either quercetin is simply binding to surface GLUT1 or its transport in and out of the cell reaches equilibrium very quickly. If quercetin is transported, the co-incubation of quercetin with other glucose inhibitors should block quercetin uptake. However, we observed that WZB-117, an exofacial binding inhibitor of GLUT1 reduced quercetin interaction, while cytochalasin B, an endofacial binding inhibitor, enhanced quercetin interaction, and BAY-876 had no effect on quercetin interaction. Taken together, these data are more consistent with quercetin simply binding to GLUT1, but not actually being transported into L929 cells via the glucose channel in GLUT1.

Caffeine inhibition of GLUT1 is dependent on the activation state of the transporter.

Caffeine has been shown to be a robust uncompetitive inhibitor of glucose uptake in erythrocytes. It preferentially binds to the nucleotide-binding site on GLUT1 in its tetrameric form and mimics the inhibitory action of ATP. Here we demonstrate that caffeine is also a dose-dependent, uncompetitive inhibitor of 2-deoxyglucose (2DG) uptake in L929 fibroblasts. The inhibitory effect on 2DG uptake in these cells was reversible with a rapid onset and was additive to the competitive inhibitory effects of glucose itself, confirming that caffeine does not interfere with glucose binding. We also report for the first time that caffeine inhibition was additive to inhibition by curcumin, suggesting distinct binding sites for curcumin and caffeine. In contrast, caffeine inhibition was not additive to that of cytochalasin B, consistent with previous data that reported that these two inhibitors have overlapping binding sites. More importantly, we show that the magnitude of maximal caffeine inhibition in L929 cells is much lower than in erythrocytes (35% compared to 90%). Two epithelial cell lines, HCLE and HK2, have both higher concentrations of GLUT1 and increased basal 2DG uptake (3-4 fold) compared to L929 cells, and subsequently display greater maximal inhibition by caffeine (66-70%). Interestingly, activation of 2DG uptake (3-fold) in L929 cells by glucose deprivation shifted the responsiveness of these cells to caffeine inhibition (35%-70%) without a change in total GLUT1 concentration. These data indicate that the inhibition of caffeine is dependent on the activity state of GLUT1, not merely on the concentration.

Curcumin directly inhibits the transport activity of GLUT1.

Curcumin, a major ingredient in turmeric, has a long history of medicinal applications in a wide array of maladies including treatment for diabetes and cancer. Seemingly counterintuitive to the documented hypoglycemic effects of curcumin, however, a recent report indicates that curcumin directly inhibits glucose uptake in adipocytes. The major glucose transporter in adipocytes is GLUT4. Therefore, this study investigates the effects of curcumin in cell lines where the major transporter is GLUT1. We report that curcumin has an immediate inhibitory effect on basal glucose uptake in L929 fibroblast cells with a maximum inhibition of 80% achieved at 75 µM curcumin. Curcumin also blocks activation of glucose uptake by azide, glucose deprivation, hydroxylamine, or phenylarsine oxide. Inhibition does not increase with exposure time and the inhibitory effects reverse within an hour. Inhibition does not appear to involve a reaction between curcumin and the thiol side chain of a cysteine residue since neither prior treatment of cells with iodoacetamide nor curcumin with cysteine alters curcumin's inhibitory effects. Curcumin is a mixed inhibitor reducing the Vmax of 2DG transport by about half with little effect on the Km. The inhibitory effects of curcumin are not additive to the effects of cytochalasin B and 75 µM curcumin actually reduces specific cytochalasin B binding by 80%. Taken together, the data suggest that curcumin binds directly to GLUT1 at a site that overlaps with the cytochalasin B binding site and thereby inhibits glucose transport. A direct inhibition of GLUT proteins in intestinal epithelial cells would likely reduce absorption of dietary glucose and contribute to a hypoglycemic effect of curcumin. Also, inhibition of GLUT1 activity might compromise cancer cells that overexpress GLUT1 and be another possible mechanism for the documented anticancer effects of curcumin.

Osthole activates glucose uptake but blocks full activation in L929 fibroblast cells, and inhibits uptake in HCLE cells.

AIMS: Osthole, a coumarin derivative, has been used in Chinese medicine and studies have suggested a potential use in treatment of diabetes and cancers. Therefore, we investigated the effects of osthole and other coumarins on GLUT1 activity in two cell lines that exclusively express GLUT1. MAIN METHODS: We measured the magnitude and time frame of the effects of osthole and related coumarins on glucose uptake in two cells lines; L929 fibroblast cells which have low GLUT1 expression levels and low basal glucose uptake and HCLE cells which have high GLUT1 concentrations and high basal uptake. We also explored the effects of these coumarins in combination with other GLUT1 activators. KEY FINDINGS: Osthole activates glucose uptake in L929 cells with a modest maximum 1.7-fold activation achieved by 50 µM with both activation and recovery occurring within minutes. However, osthole blocks full acute activation of glucose uptake by other, more robust activators. This behavior mimics the effects of other thiol reactive compounds and suggests that osthole is interacting with cysteine residues, possibly within GLUT1 itself. Coumarin, 7-hydroxycoumarin, and 7-methoxycoumarin, do not affect glucose uptake, which is consistent with the notion that the isoprenoid structure in osthole may be important to gain membrane access to GLUT1. In contrast to its effects in L929 cells, osthole inhibits basal glucose uptake in the more active HCLE cells. SIGNIFICANCE: The differential effects of osthole in L929 and HCLE cells indicated that regulation of GLUT1 varies, likely depending on its membrane concentration.

Alkaline pH activates the transport activity of GLUT1 in L929 fibroblast cells.

The widely expressed mammalian glucose transporter, GLUT1, can be acutely activated in L929 fibroblast cells by a variety of conditions, including glucose deprivation, or treatment with various respiration inhibitors. Known thiol reactive compounds including phenylarsine oxide and nitroxyl are the fastest acting stimulators of glucose uptake, implicating cysteine biochemistry as critical to the acute activation of GLUT1. In this study, we report that in L929 cells glucose uptake increases 6-fold as the pH of the uptake solution is increased from 6 to 9 with the half-maximal activation at pH 7.5; consistent with the pKa of cysteine residues. This pH effect is essentially blocked by the pretreatment of the cells with either iodoacetamide or cinnamaldehyde, compounds that form covalent adducts with reduced cysteine residues. In addition, the activation by alkaline pH is not additive at pH 8 with known thiol reactive activators such as phenylarsine oxide or hydroxylamine. Kinetic analysis in L929 cells at pH 7 and 8 indicate that alkaline conditions both increases the Vmax and decreases the Km of transport. This is consistent with the observation that pH activation is additive to methylene blue, which activates uptake by increasing the Vmax, as well as to berberine, which activates uptake by decreasing the Km. This suggests that cysteine biochemistry is utilized in both methylene blue and berberine activation of glucose uptake. In contrast a pH increase from 7 to 8 in HCLE cells does not further activate glucose uptake. HCLE cells have a 25-fold higher basal glucose uptake rate than L929 cells and the lack of a pH effect suggests that the cysteine biochemistry has already occurred in HCLE cells. The data are consistent with pH having a complex mechanism of action, but one likely mediated by cysteine biochemistry.

Differential regulation of GLUT1 activity in human corneal limbal epithelial cells and fibroblasts.

The corneal epithelial tissue is a layer of rapidly growing cells that are highly glycolytic and express GLUT1 as the major glucose transporter. It has been shown that GLUT1 in L929 fibroblast cells and other cell lines can be acutely activated by a variety agents. However, the acute regulation of glucose uptake in corneal cells has not been systematically investigated. Therefore, we examined glucose uptake in an immortalized human corneal-limbal epithelial (HCLE) cell line and compared it to glucose uptake in L929 fibroblast cells, a cell line where glucose uptake has been well characterized. We report that the expression of GLUT1 in HCLE cells is 6.6-fold higher than in L929 fibroblast cells, but the HCLE cells have a 25-fold higher basal rate of glucose uptake. Treatment with agents that interfere with mitochondrial metabolism, such as sodium azide and berberine, activate glucose uptake in L929 cells over 3-fold, but have no effect on glucose uptake HCLE cells. Also, agents known to react with thiols, such cinnamaldehyde, phenylarsine oxide and nitroxyl stimulate glucose uptake in L929 cells 3-4-fold, but actually inhibit glucose uptake in HCLE cells. These data suggest that in the fast growing HCLE cells, GLUT1 is expressed at a higher concentration and is already highly activated at basal conditions. These data support a model for the acute activation of GLUT1 that suggests that the activity of GLUT1 is enhanced by the formation of an internal disulfide bond within GLUT1 itself.

Hydroxylamine acutely activates glucose uptake in L929 fibroblast cells.

Nitroxyl (HNO) has a unique, but varied, set of biological properties including beneficial effects on cardiac contractility and stimulation of glucose uptake by GLUT1. These biological effects are largely initiated by HNO's reaction with cysteine residues of key proteins. The intracellular production of HNO has not yet been demonstrated, but the small molecule, hydroxylamine (HA), has been suggested as possible intracellular source. We examined the effects of this molecule on glucose uptake in L929 fibroblast cells. HA activates glucose uptake from 2 to 5-fold within two minutes. Prior treatment with thiol-active compounds, such as iodoacetamide (IA), cinnamaldehyde (CA), or phenylarsine oxide (PAO) blocks HA-activation of glucose uptake. Incubation of HA with the peroxidase inhibitor, sodium azide, also blocks the stimulatory effects of HA. This suggests that HA is oxidized to HNO by L929 fibroblast cells, which then reacts with cysteine residues to exert its stimulatory effects. The data suggest that GLUT1 is acutely activated in L929 cells by modification of cysteine residues, possibly the formation of a disulfide bond within GLUT1 itself.

Nitroxyl (HNO) acutely activates the glucose uptake activity of GLUT1.

Nitroxyl (HNO) is a molecule of significant interest due to its unique pharmacological properties, particularly within the cardiovascular system. A large portion of HNO biological effects can be attributed to its reactivity with protein thiols, where it can generate disulfide bonds. Evidence from studies in erythrocytes suggests that the activity of GLUT1 is enhanced by the formation of an internal disulfide bond. However, there are no reports that document the effects of HNO on glucose uptake. Therefore, we examined the acute effects of Angeli's salt (AS), a HNO donor, on glucose uptake activity of GLUT1 in L929 fibroblast cells. We report that AS stimulates glucose uptake with a maximum effective concentration of 5.0 mM. An initial 7.2-fold increase occurs within 2 min, which decreases and plateaus to a 4.0-fold activation after 10 min. About 60% of the 4.0-fold activation recovers within 10 min, and 40% remains after an hour. The activation is blocked by the pretreatment of cells with thiol-reactive compounds, iodoacetamide (0.75 mM), cinnamaldehyde (2.0 mM), and phenylarsine oxide (10 µM). The effects of AS are not additive to the stimulatory effects of other acute activators of glucose uptake in L929 cells, such as azide (5 mM), berberine (50 µM), or glucose deprivation. These data suggest that GLUT1 is acutely activated in L929 cells by the formation of a disulfide bond, likely within GLUT1 itself.

Berberine acutely activates the glucose transport activity of GLUT1.

Berberine, which has a long history of use in Chinese medicine, has recently been shown to have efficacy in the treatment of diabetes. While the hypoglycemic effect of berberine has been clearly documented in animal and cell line models, such as 3T3-L1 adipocytes and L6 myotube cells, the mechanism of action appears complex with data implicating activation of the insulin signaling pathway as well as activation of the exercise or AMP kinase-mediated pathway. There have been no reports of the acute affects of berberine on the transport activity of the insulin-insensitive glucose transporter, GLUT1. Therefore, we examined the acute effects of berberine on glucose uptake in L929 fibroblast cells, a cell line that express only GLUT1. Berberine- activated glucose uptake reaching maximum stimulation of five-fold at >40 µM. Significant activation (P < 0.05) was measured within 5 min reaching a maximum by 30 min. The berberine effect was not additive to the maximal stimulation by other known stimulants, azide, methylene blue or glucose deprivation, suggesting shared steps between berberine and these stimulants. Berberine significantly reduced the K(m) of glucose uptake from 6.7 ± 1.9 mM to 0.55 ± 0.08 mM, but had no effect on the V(max) of uptake. Compound C, an inhibitor of AMP kinase, did not affect berberine-stimulated glucose uptake, but inhibitors of downstream kinases partially blocked berberine stimulation. SB203580 (inhibitor of p38 MAP kinase) did not affect submaximal berberine activation, but did lower maximal berberine stimulation by 26%, while PD98059 (inhibitor of ERK kinase) completely blocked submaximal berberine activation and decreased the maximal stimulation by 55%. It appears from this study that a portion of the hypoglycemic effects of berberine can be attributed to its acute activation of the transport activity of GLUT1.

Effects of cinnamaldehyde on the glucose transport activity of GLUT1.

There is accumulating evidence that cinnamon extracts contain components that enhance insulin action. However, little is know about the effects of cinnamon on non-insulin stimulated glucose uptake. Therefore, the effects of cinnamaldehyde on the glucose transport activity of GLUT1 in L929 fibroblast cells were examined under both basal conditions and conditions where glucose uptake is activated by glucose deprivation. The data reveal that cinnamaldehyde has a dual action on the glucose transport activity of GLUT1. Under basal conditions it stimulates glucose uptake and reaches a 3.5 fold maximum stimulation at 2.0mM. However, cinnamaldehyde also inhibits the activation of glucose uptake by glucose deprivation in a dose dependent manner. Experiments with cinnamaldehyde analogs reveal that these activities are dependent on the α,ß-unsaturated aldehyde structural motif in cinnamaldehyde. The inhibitory, but not the stimulatory activity of cinnamaldehyde was maintained after a wash-recovery period. Pretreatment of cinnamaldehyde with thiol-containing compounds, such as ß-mercaptoethanol or cysteine, blocked the inhibitory activity of cinnamaldehyde. These results suggest that cinnamaldehyde inhibits the activation of GLUT1 by forming a covalent link to target cysteine residue/s. This dual activity of cinnamaldehyde on the transport activity of GLUT1 suggests that cinnamaldehyde is not a major contributor to the anti-diabetic properties of cinnamon.

Verapamil inhibits the glucose transport activity of GLUT1.

Calcium channel blocker toxicity has been associated with marked hyperglycemia responsive only to high-dose insulin therapy. The exact mechanism(s) of this induced hyperglycemia has not been clearly delineated. The glucose transporter GLUT1 is expressed in a wide variety of cell types and is largely responsible for a basal level of glucose transport. GLUT1 also is activated by cell stress. The specific purpose of this study was to investigate the effects of the calcium channel blocker verapamil on the glucose uptake activity of GLUT1 in L929 fibroblasts cells. Dose-dependent effects of verapamil on glucose uptake were studied using L929 fibroblast cells with 2-deoxyglucose. Verapamil had a dose-dependent inhibitory effect on both basal and stress-activated transport activity of GLUT1. Basal activity was inhibited 50% by 300 µM verapamil, while 150 µM verapamil completely inhibited the activation induced by the stress of glucose deprivation. These effects were reversible and required verapamil to be present during the stress. Alteration of calcium concentrations by addition of 5 mM CaCl2 or 4 mM EDTA had no effect on verapamil action. This study reveals the unique finding that verapamil has inhibitory effects on the transport activity of GLUT1 independent of its effects on calcium concentrations. The inhibition of GLUT1 may be one of the contributing factors to the hyperglycemia observed in CCB poisoning.

Dual action of phenylarsine oxide on the glucose transport activity of GLUT1.

An early event in the toxic effects of organic arsenic compounds, such as phenylarsine oxide (PAO), is an inhibition of glucose uptake. Glucose uptake involving the glucose transporter, GLUT4 is inhibited by PAO indicating an importance of vicinal sulfhydryls in insulin-stimulated glucose uptake. However, the data on effects of PAO on GLUT1 are conflicting. This study investigated the effects of PAO on glucose uptake in L929 fibroblast cells, cells, which express only GLUT1. The data presented here reveal a dual effect of PAO. At low concentrations or short exposure times PAO stimulated glucose uptake reaching a peak activation of about 400% at 3 microM. At higher concentrations (40 microM), PAO clearly inhibited glucose uptake. At intermediate concentrations (10 microM), PAO had no effect under basal conditions but completely inhibited activation of glucose uptake by glucose deprivation and partially inhibited methylene blue-stimulated glucose uptake. PAO increased the specific binding of cytochalasin B to GLUT1 suggesting a direct interaction with the transporter. These data are most consistent with PAO interacting with multiple proteins that regulate the activity of this transporter, one of which may be GLUT1 itself. The identity of these proteins will require further investigation.

Methyl-beta-cyclodextrin directly binds methylene blue and blocks both its cell staining and glucose uptake stimulatory effects.

GLUT1, the most ubiquitously expressed member of the GLUT family of glucose transporters, can be acutely activated by a variety of cell stresses. Methylene blue activates glucose transport activity of GLUT1 in L929 fibroblast cells presumably by a redox cycling of MB, which generates an oxidative stress. Data shown here reveal that methyl-beta-cyclodextrin (MCD) blocks both the staining of cells and activation of glucose uptake by directly binding to MB. MCD binding to MB was qualitatively demonstrated by a significantly slower dialysis rate of MB in the presence of MCD. Analysis of the complete spectra of aqueous MB solutions and MB plus MCD solutions by a factor analysis program called SIVVU indicated that these equilibria can be modeled by three species: MB monomer, MB dimer, and MCD-MB inclusion complex. The molar extinction coefficients for each species from 500 to 700nm were determined. The equilibrium association constant (K(a)) for MB dimer formation was measured at 5846+/-30M(-1) and the K(a) for formation of the MCD-MB complex was 310+/-10M(-1). MCD also dramatically enhances the destaining rate of MB-stained cells. The loss of MB from the cell is tightly correlated with the loss of activated glucose uptake. This suggests that the MB activation of glucose uptake is likely not caused by its redox cycling, but more likely the result of a specific interaction between MB and a protein directly involved in the activation of GLUT1.

Acute activation of glucose uptake by glucose deprivation in L929 fibroblast cells.

Glucose is a very important energy source for a wide variety of cells, and the ability of cells to respond to changes in glucose availability or other cell stresses is of critical importance. Many mammalian cells respond to acute stress by increasing the V(max) of transport through GLUT1; the most ubiquitously expressed glucose transporter isoform. This study investigated the acute response of glucose uptake to glucose deprivation in L929 fibroblast cells--a cell line that expresses only the GLUT1 transporter. Results indicated that glucose deprivation of only a minute activated glucose uptake 10-fold and reached a maximum of 20-fold within 10 min. The activation was dose dependent and only partially muted by addition of up to 20mM pyruvate as an alternate energy source. In contrast to the kinetics of acute metabolic stress, glucose deprivation decreased the K(m) of transport, but did not alter the V(max). Maximal activation of glucose transport by glucose deprivation was completely additive to activation of transport by methylene blue--a stimulant that increased the V(max) of transport without a change in the K(m). Glucose-deprived activation of glucose transport was not inhibited by wortmannin or herbimycin A, but was completely inhibited by phenylarsine oxide. Altogether, the data indicate that L929 fibroblast cells respond quickly and robustly to the cell stress of glucose deprivation and methylene blue treatment by two distinct activation pathways.

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.

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.