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

Kuipers, David P.; Scripture, Jared P.; Gunnink, Stephen M.; Salie, Matthew J.; Schotanus, Mark P.; Ubels, John L.; Louters, Larry L.

doi:10.1016/j.biochi.2012.09.022

Cited by 14 publications

(18 citation statements)

References 40 publications

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“…2, indicate that curcumin inhibits 2DG uptake in a dose dependent manner with a maximum of 84% inhibition at 70–100 μM with an IC 50 = 19 μM. We confirmed this inhibitory effect of curcumin in HCLE and HK2 cells, two other cell lines where GLUT1 is the primary transporter responsible for 2DG uptake [32,33]. The effects of a submax (25 μM) and maximum (75 μM) effective concentrations of curcumin were measured and reported on Table 1.…”

Section: Resultssupporting

confidence: 61%

Curcumin directly inhibits the transport activity of GLUT1

et al. 2016

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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.

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Section: Resultssupporting

confidence: 61%

Curcumin directly inhibits the transport activity of GLUT1

et al. 2016

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“…The enhanced uptake was caused by an increase expression combined with an activation of the transporter. It was observed that compounds which activated glucose uptake in L929 cells either had no effect in HCLE cell, or in the case of thiol reactive compounds, actually inhibited glucose uptake [30]. If GLUT1 in HCLE cells is already activated by involvement of the key thiols, we hypothesized that alkaline conditions would not further activate glucose uptake.…”

Section: Resultsmentioning

confidence: 97%

“…Previous work has shown that HCLE cells express GLUT1 as the primary glucose transporter and have much higher glucose uptake rates than L929 cells [30]. The enhanced uptake was caused by an increase expression combined with an activation of the transporter.…”

Section: Resultsmentioning

confidence: 99%

“…HCLE cells have a much higher concentrations of GLUT1 and glucose uptake rates and activators effective in L929 cells, including berberine, MB, and the thiol active compounds, do not further active glucose uptake [30]. This suggests that the thiol chemistry that may be initiated by the change from pH 7 to 8 has already occurred in HCLE cells.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2014

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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.

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“…Our results argue against such a mechanism because 1) they suggest that the drop in OXPHOS-mediated ATP production is fully compensated for by increased glycolytic activity, and 2) they demonstrate that total cellular ATP amount is not significantly reduced in cells treated with OXPHOS inhibitors for 24 h. Glut1 activity might also be increased by a mechanism involving AMP-activated protein kinase, activation (phosphorylation) of which was linked to stimulation of Biophysical Journal 109 (7) 1372-1386 GLC transport during OXPHOS inhibition (9,67). Alternatively, Glut1 redox modification and oligomerization were demonstrated to regulate Glut1 activity (68)(69)(70)(71) and the amount of active Glut1 at the PM might be increased by stimulation of Glut1 translocation from internal storage vesicles to the PM or by activation of PM-localized Glut1 by OXPHOS inhibition (9)(10)(11)13).…”

Section: Oxphos Inhibition Stimulates Cellular Glc Uptakementioning

confidence: 99%

Mitoenergetic Dysfunction Triggers a Rapid Compensatory Increase in Steady-State Glucose Flux

Liemburg-Apers

Schirris

Rüssel

et al. 2015

Biophysical Journal

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ATP can be produced in the cytosol by glycolytic conversion of glucose (GLC) into pyruvate. The latter can be metabolized into lactate, which is released by the cell, or taken up by mitochondria to fuel ATP production by the tricarboxylic acid cycle and oxidative phosphorylation (OXPHOS) system. Altering the balance between glycolytic and mitochondrial ATP generation is crucial for cell survival during mitoenergetic dysfunction, which is observed in a large variety of human disorders including cancer. To gain insight into the kinetic properties of this adaptive mechanism we determined here how acute (30 min) inhibition of OXPHOS affected cytosolic GLC homeostasis. GLC dynamics were analyzed in single living C2C12 myoblasts expressing the fluorescent biosensor FLII(12)Pglu-700μδ6 (FLII). Following in situ FLII calibration, the kinetic properties of GLC uptake (V1) and GLC consumption (V2) were determined independently and used to construct a minimal mathematical model of cytosolic GLC dynamics. After validating the model, it was applied to quantitatively predict V1 and V2 at steady-state (i.e., when V1 = V2 = Vsteady-state) in the absence and presence of OXPHOS inhibitors. Integrating model predictions with experimental data on lactate production, cell volume, and O2 consumption revealed that glycolysis and mitochondria equally contribute to cellular ATP production in control myoblasts. Inhibition of OXPHOS induced a twofold increase in Vsteady-state and glycolytic ATP production flux. Both in the absence and presence of OXPHOS inhibitors, GLC was consumed at near maximal rates, meaning that GLC consumption is rate-limiting under steady-state conditions. Taken together, we demonstrate here that OXPHOS inhibition increases steady-state GLC uptake and consumption in C2C12 myoblasts. This activation fully compensates for the reduction in mitochondrial ATP production, thereby maintaining the balance between cellular ATP supply and demand.

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

Cited by 14 publications

References 40 publications

Curcumin directly inhibits the transport activity of GLUT1

Curcumin directly inhibits the transport activity of GLUT1

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

Mitoenergetic Dysfunction Triggers a Rapid Compensatory Increase in Steady-State Glucose Flux

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