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. 2015 May 15;308(10):C827-34.
doi: 10.1152/ajpcell.00001.2015. Epub 2015 Feb 25.

Caffeine inhibits glucose transport by binding at the GLUT1 nucleotide-binding site

Jay M Sage  1 , Anthony J Cura  2 , Kenneth P Lloyd  1 , Anthony Carruthers  3
Affiliations

Caffeine inhibits glucose transport by binding at the GLUT1 nucleotide-binding site

Jay M Sage et al. Am J Physiol Cell Physiol. .

Abstract

Glucose transporter 1 (GLUT1) is the primary glucose transport protein of the cardiovascular system and astroglia. A recent study proposes that caffeine uncompetitive inhibition of GLUT1 results from interactions at an exofacial GLUT1 site. Intracellular ATP is also an uncompetitive GLUT1 inhibitor and shares structural similarities with caffeine, suggesting that caffeine acts at the previously characterized endofacial GLUT1 nucleotide-binding site. We tested this by confirming that caffeine uncompetitively inhibits GLUT1-mediated 3-O-methylglucose uptake in human erythrocytes [Vmax and Km for transport are reduced fourfold; Ki(app) = 3.5 mM caffeine]. ATP and AMP antagonize caffeine inhibition of 3-O-methylglucose uptake in erythrocyte ghosts by increasing Ki(app) for caffeine inhibition of transport from 0.9 ± 0.3 mM in the absence of intracellular nucleotides to 2.6 ± 0.6 and 2.4 ± 0.5 mM in the presence of 5 mM intracellular ATP or AMP, respectively. Extracellular ATP has no effect on sugar uptake or its inhibition by caffeine. Caffeine and ATP displace the fluorescent ATP derivative, trinitrophenyl-ATP, from the GLUT1 nucleotide-binding site, but d-glucose and the transport inhibitor cytochalasin B do not. Caffeine, but not ATP, inhibits cytochalasin B binding to GLUT1. Like ATP, caffeine renders the GLUT1 carboxy-terminus less accessible to peptide-directed antibodies, but cytochalasin B and d-glucose do not. These results suggest that the caffeine-binding site bridges two nonoverlapping GLUT1 endofacial sites-the regulatory, nucleotide-binding site and the cytochalasin B-binding site. Caffeine binding to GLUT1 mimics the action of ATP but not cytochalasin B on sugar transport. Molecular docking studies support this hypothesis.

Keywords: ATP; GLUT1; caffeine; erythrocyte; glucose transport.

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Figures

Fig. 1.
Fig. 1.
A: concentration dependence of caffeine inhibition of 100 μM 3-O-methylglucose (3-OMG) uptake. Ordinate: rate of sugar uptake in μmol·l cell water−1·min−1; abscissa: caffeine concentration in the uptake medium in mM. Results are shown as means ± SE for at least 4 measurements made in duplicate. The curve drawn through the points was computed by nonlinear regression assuming that saturable inhibition of transport is described by Eq. 2. The results are: v0 = 16.7 ± 0.2 μmol·l−1·min−1; Vi = 18.1 ± 0.3 μmol·l−1·min−1; apparent inhibition constant Ki(app) = 3.5 ± 0.2 mM, R2 = 0.99. B: caffeine is an uncompetitive inhibitor of 3-OMG uptake by human red blood cells (RBCs). Ordinate: rate of sugar uptake in μmol·l cell water−1·min−1; abscissa: extracellular 3-OMG concentration in mM. Results are shown as means ± SE for at least 4 measurements made in duplicate with control cells (○) and for cells exposed to 5 mM caffeine (●). Control experiments indicate that results obtained by preincubating cells with caffeine for 15 min are indistinguishable from the those obtained by simply adding caffeine during the 30 s of sugar uptake measurement. The curves drawn through the points were computed by nonlinear regression assuming that transport is described by Michaelis-Menten kinetics (Eq. 1). The results are: control, Vmax = 151 ± 12 μmol·l−1·min−1; Km = 1.3 ± 0.3 mM, R2 = 0.87; Caffeine, Vmax = 32 ± 2 μmol·l−1·min−1; Km = 0.4 ± 0.1 mM, R2 = 0.80.
Fig. 2.
Fig. 2.
Dixon plot of caffeine inhibition of 3-OMG uptake in RBC ghosts. Ordinate: 1/sugar uptake (min·mol−1·l−1); Abscissa: caffeine concentration in mM. Results are shown as means ± SE for at least 3 measurements made in duplicate and are shown for control ghosts (○), ghosts containing 4 mM ATP (●), ghosts containing 4 mM AMP (△), and nucleotide-free, resealed ghosts exposed to 4 mM extracellular ATP (▲). The lines drawn through the points were computed by linear regression and have the following constants: control slope = 337.0 ± 44.9 min, x-intercept = −0.91 ± 0.34 mM, y-intercept = 305,226 ± 111,768 min/M, R2 = 0.72; 4 mM intracellular ATP slope = 44.5 ± 4.6 min, x-intercept = −2.58 ± 0.55 mM, y-intercept = 111,949 ± 10,641 min/M, R2 = 0.83; 4 mM intracellular AMP slope = 131.8 ± 11.1 min, x-intercept = −2.41 ± 0.45 mM, y-intercept = 318,946 ± 27,465 min/M, R2 = 0.85; 4 mM extracellular ATP slope = 354.6 ± 56.7 min, x-intercept = −0.96 ± 0.63 mM, y-intercept = 340,677 ± 135,181 min/M, R2 = 0.67. In all instances the slope is significantly different from zero (P < 0.0001).
Fig. 3.
Fig. 3.
Caffeine displaces ATP from glucose transporter 1 (GLUT1). A: emission spectra of 5 μM trinitrophenyl-ATP (TNP-ATP) in the absence (dashed lines) or presence (continuous lines) of 100 nM GLUT1. Suspensions were excited at 408 nm, and the resulting emission (ordinate) was measured over 500–600 nm (abscissa). Results are shown for TNP-ATP emission in the absence of added ligands (black lines) and for emission in the presence of 5 mM d-glucose (purple lines), 10 μM cytochalasin B (CB; blue lines), 5 mM ATP (red lines), or 5 mM caffeine (green lines). B: GLUT1-specific TNP-ATP maximum fluorescence shown in the absence or presence of ligands. Ordinate: fluorescence; abscissa: ligand additions. Results are shown as means ± SE for 3 separate measurements. *Measured emission is significantly less than that observed in the absence of added ligand (P ≤ 0.037, 1-tailed, paired t-test).
Fig. 4.
Fig. 4.
Caffeine and ATP promote conformational change in the GLUT1 COOH terminus. Ordinate: COOH-terminal antibody (C-Ab) binding to GLUT1 measured by ELISA (relative antibody binding, %); abscissa: C-Ab binding conditions. C-Ab binding to unsealed GLUT1 proteoliposomes was measured in the presence and absence of ATP (5–20 mM, gray bars), caffeine (5–20 mM, diagonal bars), d-glucose (d-Glc, 5–20 mM, black bars), or CB (5–20 μM, hatched bars). The numbers below the chart indicate the concentration of ligand present during the binding assay. Results are shown as means ± SE of 3–16 measurements made in duplicate. *Unpaired, 1-tailed t-test analysis indicates that the results are significantly less than that observed in control antibody binding (P ≤ 0.0027).
Fig. 5.
Fig. 5.
Docking analysis of caffeine, ATP, and CB binding to GLUT1. A: GLUT1 crystal structure (28) showing ATP, caffeine, and CB located in their respective, highest affinity docking sites. The images were generated in PyMol and show GLUT1 in cartoon representation. ATP (red), caffeine (blue), and CB (yellow) are shown as space-filling representations. GLUT1 transmembrane helix (TM) 5 is eliminated to reveal the putative ligand binding sites. TMs 8 and 9, which have previously been demonstrated to be the site of azidoATP photo-incorporation (44), are indicated in red. TMs 1, 3, 6, 8, 9, and 10 are numbered. The approximate margins of the lipid bilayer are indicated by the horizontal lines. Residues coordinating ATP binding (and their TM locations) are: F26 (TM1), Q161 (TM5), Q282 and Q283 (TM7), E380 and P385 (TM10), N411 and N415 (TM11). The computed ΔG for ATP binding is −8.5 kcal/mol. Residues coordinating caffeine binding (and their TM locations) are: Q161 (TM5) and P385 (TM10). The computed ΔG for ATP binding is −5.7 kcal/mol. Residues coordinating CB binding (and their TM and loop locations) are: V83 (TM2), M142 (TM4), W388, F389 and A392 (TM10 and loop 10–11), I404 and G408 (TM 11). The computed ΔG for CB binding is −10.3 kcal/mol. B: space-filling models of ATP (red) and caffeine (blue) and their corresponding stick models docked in their respective GLUT1-binding sites. C: space-filling models of caffeine (blue) and CB (yellow) and their corresponding stick models docked in their respective GLUT1-binding sites. D: space-filling models of ATP (red) and CB (yellow) and their corresponding stick models docked in their respective GLUT1-binding sites.
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References

    1. Afzal I, Cunningham P, Naftalin RJ. Interactions of ATP, oestradiol, genistein and the anti-oestrogens, faslodex (ICI 182780) and tamoxifen, with the human erythrocyte glucose transporter, GLUT1. Biochem J 365: 707–719, 2002. - PMC - PubMed
    1. Armstrong CM, Bezanilla F. Inactivation of the sodium channel. II. Gating current experiments. J Gen Physiol 70: 567–590, 1977. - PMC - PubMed
    1. Ayala FR, Rocha RM, Carvalho KC, Carvalho AL, da Cunha IW, Lourenço SV, Soares FA. GLUT1 and GLUT3 as potential prognostic markers for Oral Squamous Cell Carcinoma. Molecules 15: 2374–2387, 2010. - PMC - PubMed
    1. Baker GF, Widdas WF. The asymmetry of the facilitated transfer system for hexoses in human red cells and the simple kinetics of a two component model. J Physiol 231: 143–165, 1973. - PMC - PubMed
    1. Baker GF, Naftalin RJ. Evidence of multiple operational affinities for d-glucose inside the human erythrocyte membrane. Biochim Biophys Acta 550: 474–484, 1979. - PubMed

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