Multiple Interactions of Glucose with the Extra-Membranous Loops of GLUT1 Aid Transport.

Molecular dynamics simulations amounting to ≈8 µs demonstrate that the glucose transporter GLUT1 undergoes structural fluctuations mediated by the fluidity of the lipid bilayer and the proximity to glucose. The fluctuations of GLUT1 increase as the glucose concentration is raised. These fluctuations are more pronounced when the lipid bilayer is in the fluid compared to the gel phase. Glucose interactions are confined to the extra-membranous residues when the lipid is in the gel phase but diffuses into the transmembrane regions in the fluid phase. Proximity of glucose to GLUT1 causes asynchronous expansions of key bottlenecks at the internal and external openings of the central pore. This is accomplished only by small conformational changes at the single residue level that lower the resistance to glucose movements, thereby permitting unsteered glucose and water movements along the entire length of the pore. When glucose is near salt bridges located at the external and internal openings of the central pore, the distance separating the polar amino acid residues guarding these apertures tends to increase in both fluid and gel phases. It is evident that the multiplicity of glucose interactions, obtained with high concentrations, amplifies the structural fluctuations in GLUT1. The findings that most of the salt bridges and the bottlenecks appear to be operated by glucose proximity suggest that the main triggers to activation of transport are located within the solvent accessible linker regions in the extramembranous zones.

UVA irradiation increases ferrous iron release from human skin fibroblast and endothelial cell ferritin: Consequences for cell senescence and aging.

UVA irradiation of human dermal fibroblasts and endothelial cells induces an immediate transient increase in cytosolic Fe(II), as monitored by the fluorescence Fe(II) reporters, FeRhonox1 in cytosol and MitoFerroGreen in mitochondria. Both superoxide dismutase (SOD) inhibition by tetrathiomolybdate (ATM) and catalase inhibition by 3-amino-1, 2, 4-triazole (ATZ) increase and prolong the cytosolic Fe(II) signal after UVA irradiation. SOD inhibition with ATM also increases mitochondrial Fe(II). Thus, mitochondria do not source the UV-dependent increase in cytosolic Fe(II), but instead reflect and amplify raised cytosolic labile Fe(II) concentration. Hence control of cytosolic ferritin iron release is key to preventing UVA-induced inflammation. UVA irradiation also increases dermal endothelial cell H2O2, as monitored by the adenovirus vector Hyper-DAAO-NES(HyPer). These UVA-dependent changes in intracellular Fe(II) and H2O2 are mirrored by increases in cell superoxide, monitored with the luminescence probe L-012. UV-dependent increases in cytosolic Fe(II), H2O2 and L-012 chemiluminescence are prevented by ZnCl2 (10 µM), an effective inhibitor of Fe(II) transport via ferritin's 3-fold channels. Quercetin (10 µM), a potent membrane permeable Fe(II) chelator, abolishes the cytosolic UVA-dependent FeRhonox1, Fe(II) and HyPer, H2O2 and increase in MitoFerroGreen Fe(II) signals. The time course of the quercetin-dependent decrease in endothelial H2O2 correlates with the decrease in FeRhox1 signal and both signals are fully suppressed by preloading cells with ZnCl2. These results confirm that antioxidant enzyme activity is the key factor in controlling intracellular iron levels, and hence maintenance of cell antioxidant capacity is vitally important in prevention of skin aging and inflammation initiated by labile iron and UVA.

Ascorbate and ferritin interactions: Consequences for iron release in vitro and in vivo and implications for inflammation.

This review discusses the chemical mechanisms of ascorbate-dependent reduction and solubilization of ferritin's ferric iron core and subsequent release of ferrous iron. The process is accelerated by low concentrations of Fe(II) that increase ferritin's intrinsic ascorbate oxidase activity, hence increasing the rate of ascorbate radical formation. These increased rates of ascorbate oxidation provide reducing equivalents (electrons) to ferritin's core and speed the core reduction rates with subsequent solubilization and release of Fe(II). Ascorbate-dependent solubilization of ferritin's iron core has consequences relating to the interpretation of 59Fe uptake sourced from 59Fe-lebelled holotransferrin into ferritin. Ascorbate-dependent reduction of the ferritin core iron solubility increases the size of ferritin's iron exchangeable pool and hence the rate and amount of exchange uptake of 59Fe into ferritin, whilst simultaneously increasing net iron release rate from ferritin. This may rationalize the inconsistency that ascorbate apparently stabilizes 59Fe ferritin and retards lysosomal ferritinolysis and whole cell 59Fe release, whilst paradoxically increasing the rate of net iron release from ferritin. This capacity of ascorbate and iron to synergise ferritin iron release has pathological significance, as it lowers the concentration at which ascorbate activates ferritin's iron release to within the physiological range (50-250 µM). These effects have relevance to inflammatory pathology and to the pro-oxidant effects of ascorbate in cancer therapy and cell death by ferroptosis.

Labile iron potentiates ascorbate-dependent reduction and mobilization of ferritin iron.

Ascorbate mobilizes iron from equine spleen ferritin by two separate processes. Ascorbate alone mobilizes ferritin iron with an apparent Km (ascorbate) ≈1.5mM. Labile iron >2µM, complexed with citrate (10mM), synergises ascorbate-dependent iron mobilization by decreasing the apparent Km (ascorbate) to ≈270µM and raising maximal mobilization rate by ≈5-fold. Catalase reduces the apparent Km(ascorbate) for both ascorbate and ascorbate+iron dependent mobilization by ≈80%. Iron mobilization by ascorbate alone has a higher activation energy (Ea=45.0±5.5kJ/mole) than when mediated by ascorbate with labile iron (10µM) (Ea=13.7±2.2kJ/mole); also mobilization by iron-ascorbate has a three-fold higher pH sensitivity (pH range 6.0-8.0) than with ascorbate alone. Hydrogen peroxide inhibits ascorbate's iron mobilizing action. EPR and autochemiluminescence studies show that ascorbate and labile iron within ferritin enhances radical formation, whereas ascorbate alone produces negligible radicals. These findings suggest that iron catalysed single electron transfer reactions from ascorbate, involving ascorbate or superoxide and possibly ferroxidase tyrosine radicals, accelerate iron mobilization from the ferroxidase centre more than EPR silent, bi-dentate two-electron transfers. These differing modes of electron transference from ascorbate mirror the known mono and bidentate oxidation reactions of dioxygen and hydrogen peroxide with di-ferrous iron at the ferroxidase centre. This study implies that labile iron, at physiological pH, complexed with citrate, synergises iron mobilization from ferritin by ascorbate (50-4000µM). This autocatalytic process can exacerbate oxidative stress in ferritin-containing inflamed tissue.

Membrane Phase-Dependent Occlusion of Intramolecular GLUT1 Cavities Demonstrated by Simulations.

Experimental evidence has shown a close correlation between the composition and physical state of the membrane bilayer and glucose transport activity via the glucose transporter GLUT1. Cooling alters the membrane lipids from the fluid to gel phase, and also causes a large decrease in the net glucose transport rate. The goal of this study is to investigate how the physical phase of the membrane alters glucose transporter structural dynamics using molecular-dynamics simulations. Simulations from an initial fluid to gel phase reduce the size of the cavities and tunnels traversing the protein and connecting the external regions of the transporter and the central binding site. These effects can be ascribed solely to membrane structural changes since in silico cooling of the membrane alone, while maintaining the higher protein temperature, shows protein structural and dynamic changes very similar to those observed with uniform cooling. These results demonstrate that the protein structure is sensitive to the membrane phase, and have implications for how transmembrane protein structures respond to their physical environment.

A computer model simulating human glucose absorption and metabolism in health and metabolic disease states.

A computer model designed to simulate integrated glucose-dependent changes in splanchnic blood flow with small intestinal glucose absorption, hormonal and incretin circulation and hepatic and systemic metabolism in health and metabolic diseases e.g. non-alcoholic fatty liver disease, (NAFLD), non-alcoholic steatohepatitis, (NASH) and type 2 diabetes mellitus, (T2DM) demonstrates how when glucagon-like peptide-1, (GLP-1) is synchronously released into the splanchnic blood during intestinal glucose absorption, it stimulates superior mesenteric arterial (SMA) blood flow and by increasing passive intestinal glucose absorption, harmonizes absorption with its distribution and metabolism. GLP-1 also synergises insulin-dependent net hepatic glucose uptake (NHGU). When GLP-1 secretion is deficient post-prandial SMA blood flow is not increased and as NHGU is also reduced, hyperglycaemia follows. Portal venous glucose concentration is also raised, thereby retarding the passive component of intestinal glucose absorption.   Increased pre-hepatic sinusoidal resistance combined with portal hypertension leading to opening of intrahepatic portosystemic collateral vessels are NASH-related mechanical defects that alter the balance between splanchnic and systemic distributions of glucose, hormones and incretins.The model reveals the latent contribution of portosystemic shunting in development of metabolic disease. This diverts splanchnic blood content away from the hepatic sinuses to the systemic circulation, particularly during the glucose absorptive phase of digestion, resulting in inappropriate increases in insulin-dependent systemic glucose metabolism.  This hastens onset of hypoglycaemia and thence hyperglucagonaemia. The model reveals that low rates of GLP-1 secretion, frequently associated with T2DM and NASH, may be also be caused by splanchnic hypoglycaemia, rather than to intrinsic loss of incretin secretory capacity. These findings may have therapeutic implications on GLP-1 agonist or glucagon antagonist usage.

Vasopressin regulation of epithelial colonic proliferation and permeability is mediated by pericryptal platelet-derived growth factor A.

Arginine vasopressin (AVP) has trophic effects on the rat distal colon, increasing the growth of pericryptal myofibroblasts and reducing the colonic crypt wall permeability. This study aimed to reproduce in vitro the effects of AVP observed in vivo using cultures of human CCD-18Co myofibroblasts and T84 colonic epithelial cells. Proliferation of myofibroblasts was quantified by bromodeoxyuridine incorporation; the expression of platelet-derived growth factor A (PDGFA), platelet-derived growth factor B, epidermal growth factor, transforming growth factor-ß and vascular endothelial growth factor was measured by PCR and the expression of epithelial junction proteins by Western blot. Arginine vasopressin stimulated myofibroblast proliferation and the expression of PDGFA without affecting the expression of platelet-derived growth factor B, epidermal growth factor, transforming growth factor-ß or vascular endothelial growth factor. These effects were prevented when AVP receptor inhibitors were present in the medium. Pre-incubation of CCD-18Co cells with anti-PDGF antibody or with an inhibitor of the PDGF receptor abolished the effects of AVP. When colonocytes were incubated with medium obtained from myofibroblasts incubated with AVP, both cell proliferation and the expression of epithelial junction proteins increased; however, direct incubation of colonocytes with AVP did not modify these variables. These results demonstrate that AVP stimulates myofibroblast proliferation and induces PDGFA secretion, implying that PDGFA mediates local myofibroblast proliferation by an autocrine feedback loop and regulates epithelial proliferation and permeability by a paracrine mechanism.

Reptation-induced coalescence of tunnels and cavities in Escherichia Coli XylE transporter conformers accounts for facilitated diffusion.

Structural changes and xylose docking to eight conformers of Escherichia Coli XylE, a xylose transporter similar to mammalian passive glucose transporters GLUTs, have been examined. Xylose docks to inward and outward facing conformers at a high affinity central site (K(i) 4-20 µM), previously identified by crystallography and additionally consistently docks to lower affinity sites in the external and internal vestibules (K(i) 12-50 µM). All these sites lie within intramolecular tunnels and cavities. Several local regions in the central transmembrane zone have large positional divergences of both skeleton carbon Cα positions and side chains. One such in TM 10 is the destabilizing sequence G388-P389-V390-C391 with an average RMSD (4.5 ± 0.4 Å). Interchange between conformer poses results in coalescence of tunnels with adjacent cavities, thereby producing a transitory channel spanning the entire transporter. A fully open channel exists in one inward-facing apo-conformer, (PDB 4ja4c) as demonstrated by several different tunnel-finding algorithms. The conformer interchanges produce a gated network within a branched central channel that permits staged ligand diffusion across the transporter during the open gate periods. Simulation of this model demonstrates that small-scale conformational changes required for sequentially opening gate with frequencies in the ns-µs time domain accommodate diffusive ligand flow between adjacent sites with association-dissociation rates in the µs-ms domain without imposing delays. This current model helps to unify the apparently opposing concepts of alternate access and multisite models of ligand transport.

Does apical membrane GLUT2 have a role in intestinal glucose uptake?

It has been proposed that the non-saturable component of intestinal glucose absorption, apparent following prolonged exposure to high intraluminal glucose concentrations, is mediated via the low affinity glucose and fructose transporter, GLUT2, upregulated within the small intestinal apical border. The evidence that the non-saturable transport component is mediated via an apical membrane sugar transporter is that it is inhibited by phloretin, after exposure to phloridzin. Since the other apical membrane sugar transporter, GLUT5, is insensitive to inhibition by either cytochalasin B, or phloretin, GLUT2 was deduced to be the low affinity sugar transport route. As in its uninhibited state, polarized intestinal glucose absorption depends both on coupled entry of glucose and sodium across the brush border membrane and on the enterocyte cytosolic glucose concentration exceeding that in both luminal and submucosal interstitial fluids, upregulation of GLUT2 within the intestinal brush border will usually stimulate downhill glucose reflux to the intestinal lumen from the enterocytes; thereby reducing, rather than enhancing net glucose absorption across the luminal surface. These states are simulated with a computer model generating solutions to the differential equations for glucose, Na and water flows between luminal, cell, interstitial and capillary compartments. The model demonstrates that uphill glucose transport via SGLT1 into enterocytes, when short-circuited by any passive glucose carrier in the apical membrane, such as GLUT2, will reduce transcellular glucose absorption and thereby lead to increased paracellular flow. The model also illustrates that apical GLUT2 may usefully act as an osmoregulator to prevent excessive enterocyte volume change with altered luminal glucose concentrations.

Implications of aberrant temperature-sensitive glucose transport via the glucose transporter deficiency mutant (GLUT1DS) T295M for the alternate-access and fixed-site transport models.

In silico glucose docking to the transporter GLUT1 templated to the crystal structure of Escherichia coli XylE, a bacterial homolog of GLUT1-4 (4GBZ.pdb), reveals multiple docking sites. One site in the external vestibule in the exofacial linker between TM7 and -8 is adjacent to a missense T295M and a 4-mer insertion mutation. Glucose docking to the adjacent site is occluded in these mutants. These mutants cause an atypical form of glucose transport deficiency syndrome (GLUT1DS), where transport into the brain is deficient, although unusually transport into erythrocytes at 4 °C appears normal. A model in which glucose traverses the transporter via a network of saturable fixed sites simulates the temperature sensitivity of normal and mutant glucose influx and the mutation-dependent alterations of influx and efflux asymmetry when expressed in Xenopus oocytes at 37 °C. The explanation for the temperature sensitivity is that at 4 °C glucose influx between the external and internal vestibules is slow and causes glucose to accumulate in the external vestibule. This retards net glucose uptake from the external solution via two parallel sites into the external vestibule, consequently masking any transport defect at either one of these sites. At 37 °C glucose transit between the external and internal vestibules is rapid, with no significant glucose buildup in the external vestibule, and thereby unmasks any transport defect at one of the parallel input sites. Monitoring glucose transport in patients' erythrocytes at higher temperatures may improve the diagnostic accuracy of the functional test of GLUT1DS.

Aldosterone induces myofibroblast EGF secretion to regulate epithelial colonic permeability.

In vivo studies show that raised aldosterone (Aldo) during low-Na adaptation regulates the growth of pericryptal myofibroblasts and reduces the permeability of the colonic epithelium. The aim of this study was to reproduce in vitro the in vivo condition of increased Aldo using human CCD-18Co myofibroblasts and T84 colonic epithelial cells to measure myofibroblast and epithelial proliferation and the expression of intercellular junction proteins. Proliferation was quantified by measuring 5-bromo-2'-deoxyuridine incorporation. The myofibroblast expression of EGF, VEGFa, and transforming growth factor-ß1 (TGF-ß1) was measured by real-time PCR and the expression of junctional complex proteins by Western blot. Aldo stimulated the proliferation of myofibroblasts by 70% (P < 0.05) and increased EGF mRNA expression by 30% (P < 0.05) without affecting VEGFa and TGF-ß1. EGF concentration in the incubation medium increased by 30% (P < 0.05) 24 h after Aldo addition, and these effects were prevented by the addition of spironolactone. Myofibroblast proliferation in response to Aldo was mediated by EGF receptor (EGFR) and involved both MAPKK and phosphatidylinositol 3-kinase pathways. When T84 cells were incubated with medium from myofibroblasts stimulated with Aldo (conditioned medium), the expression of ß-catenin and claudin IV was increased by 30% (P < 0.05) and proliferation by 40% (P < 0.05). T84 proliferation decreased when α-EGF, or the EGFR antagonist AG1478, was present. Results in vivo indicate that rats fed a low-salt diet showed an increased expression of EGF and EGFR in the colonic mucosa. These results support the view that changes in colonic permeability during low-Na adaptation are mediated by the EGF secreted by myofibroblasts in response to raised Aldo.

Quercetin-iron chelates are transported via glucose transporters.

Flavonoids are well-known antioxidants and free radical scavengers. Their metal-binding activity suggests that they could be effective protective agents in pathological conditions caused by both extracellular and intracellular oxidative stress linked to metal overload. Quercetin is both a permeant ligand via glucose transport proteins (GLUTs) and a high-affinity inhibitor of GLUT-mediated glucose transport. Chelatable "free iron" at micromolar concentrations in body fluids is a catalyst of hydroxyl radical (OH(•)) production from hydrogen peroxide. A number of flavonoids, e.g., quercetin, luteolin, chrysin, and 3,6-dihydroxyflavone, have been demonstrated to chelate intracellular iron and suppress OH(•) radical production in Madin Darby canine kidney cells. The most effective chelation comes from the flavonone B ring catechol found in both quercetin and luteolin. We show here that quercetin concentrations of <1µM can facilitate chelatable iron shuttling via GLUT1 in either direction across the cell membrane. These siderophoric effects are inhibited by raised quercetin concentrations (>1µM) or GLUT inhibitors, e.g., phloretin or cytochalasin B, and iron efflux is enhanced by impermeant extracellular iron chelators, either desferrioxamine or rutin. This iron shuttling property of quercetin might be usefully harnessed in chelotherapy of iron-overload conditions.

Reassessment of models of facilitated transport and cotransport.

Most membrane transport models are determinate, requiring the transported ligand(s) to bind initially to a vacant site, which undergoes translation and releases ligand to the alternate side. The carrier reverts to its initial position to complete the net transport cycle. Ligand affinity may change during translation, but this must be compensated by an equivalent energy change(s) within the transport cycle. However, any asymmetric cyclic equilibrium deduced on this basis is thermodynamically fallacious. Determinate cotransport models imply lossless stoichiometric relationships between the complexed cotransported ligands. Independent ligand leakage apart from the mobile cotransport complex must occur outside the canonical cotransport pathway. In contrast, stochastic transport models assume independent ligand diffusion through a variably occluded channel(s) containing binding sites where ligands may undergo bimolecular exchanges. Energy dissipation is intrinsic to all stochastic transport models and occurs within the primary transport pathway. Frictional interactions within a shared path generate flow coupling between ligands. The primary driving forces causing transmembrane ligand flows are their electrochemical potential differences between the external solutions. Demonstrations that ligand exchanges in CLC and neurotransmitter transporters can be multimodal, encompassing both "channel"-like high and "transporter"-like lower conductance states and have independently regulated import and export exchange fluxes are major challenges to determinate models but are explicable by transient widening of a close-encounter region within the channel, leading to decreased coupling and enhanced efflux.

Quercetin is a substrate for the transmembrane oxidoreductase Dcytb.

Duodenal cytochrome b (Dcytb) is a transmembrane oxidoreductase protein found in apical membranes of duodenal enterocytes, as well as human erythrocytes, with the capacity to transport electrons donated by cytosolic ascorbate to extracellular electron receptors such as Fe(III), dehydroascorbate, or molecular O(2). We have investigated the capacity of the flavonoid quercetin to act as an electron donor for Dcytb in a manner similar to that of ascorbate by observing the reduction of extracellular Fe(III) to Fe(II) in either Madin-Darby canine kidney (MDCK) cells overexpressing Dcytb (Dcytb(+)) or Dcytb-null MDCK cells. In Dcytb(+) cells there is a saturable increase in extracellular Fe(III) reduction in response to increasing intracellular quercetin concentrations (K(m)=6.53+/-1.57 microM), in addition to a small linear response, whereas in Dcytb-null cells there is only a small linear increase in extracellular Fe(III) reduction. No extracellular Fe(III) reduction occurs in Dcytb-null cells when the cells are preloaded with ascorbate. Flavonoids such as quercetin at their physiological concentrations can therefore function as modulators of ferric reductases, enhancing the import of Fe(II) and also providing extracellular reducing potential.

Comparison of effects of green tea catechins on apicomplexan hexose transporters and mammalian orthologues.

Here we have investigated the inhibitory properties of green tea catechins on the Plasmodium falciparum hexose transporter (PfHT), the Babesia bovis hexose transporter 1 (BboHT1) and the mammalian facilitative glucose transporters, GLUT1 and GLUT5, expressed in Xenopus laevis oocytes. (-)-Epicatechin-gallate (ECG) and (-)-epigallocatechin-gallate (EGCG) inhibited D-glucose transport by GLUT1 and PfHT, and D-fructose transport by GLUT5, with apparent K(i) values between 45 and 117 microM. BboHT1 was more potently inhibited by the ungallated catechins (-)-epicatechin (EC) and (-)-epigallocatechin (EGC), with apparent K(i) values of 108 and 168 microM, respectively. Site-directed mutagenesis experiments provided little further support for previously reported models of catechin binding to hexose transporters. Furthermore, P. falciparum growth inhibition by catechins was not affected by the external D-glucose concentration. Our results provide new data on the inhibitory action of catechins against sugar transporters but were unable to elucidate the antimalarial mechanism of action of these agents.

Alternating carrier models of asymmetric glucose transport violate the energy conservation laws.

Alternating access transporters with high-affinity externally facing sites and low-affinity internal sites relate substrate transit directly to the unliganded asymmetric "carrier" (Ci) distribution. When both bathing solutions contain equimolar concentrations of ligand, zero net flow of the substrate-carrier complex requires a higher proportion of unliganded low-affinity inside sites (proportional, variant 1/KD(in)) and slower unliganded "free" carrier transit from inside to outside than in the reverse direction. However, asymmetric rates of unliganded carrier movement, kij, imply that an energy source, DeltaGcarrier = RT ln (koi/kio) = RT ln (Cin/Cout) = RT ln (KD(in)/KD(out)), where R is the universal gas constant (8.314 Joules/M/K degrees), and T is the temperature, assumed here to be 300 K degrees , sustains the asymmetry. Without this invalid assumption, the constraints of carrier path cyclicity, combined with asymmetric ligand affinities and equimolarity at equilibrium, are irreconcilable, and any passive asymmetric uniporter or cotransporter model system, e.g., Na-glucose cotransporters, espousing this fundamental error is untenable. With glucose transport via GLUT1, the higher maximal rate and Km of net ligand exit compared to net ligand entry is only properly simulated if ligand transit occurs by serial dissociation-association reactions between external high-affinity and internal low-affinity immobile sites. Faster intersite transit rates occur from lower-affinity sites than from higher-affinity sites and require no other energy source to maintain equilibrium. Similar constraints must apply to cotransport.