Red wine and green tea flavonoids are cis-allosteric activators and competitive inhibitors of glucose transporter 1 (GLUT1)-mediated sugar uptake.

The antioxidant- and flavonoid-rich contents of red wine and green tea are reported to offer protection against cancer, cardiovascular disease, and diabetes. Some studies, however, show that flavonoids inhibit GLUT1-mediated, facilitative glucose transport, raising the possibility that their interaction with GLUT1 and subsequent downstream effects on carbohydrate metabolism may also impact health. The present study explores the structure-function relationships of flavonoid-GLUT1 interactions. We find that low concentrations of flavonoids act as cis-allosteric activators of sugar uptake, whereas higher concentrations competitively inhibit sugar uptake and noncompetitively inhibit sugar exit. Studies with heterologously expressed human GLUT1, -3, or -4 reveal that quercetin-GLUT1 and -GLUT4 interactions are stronger than quercetin-GLUT3 interactions, that epicatechin gallate (ECG) is more selective for GLUT1, and that epigallocatechin gallate (EGCG) is less GLUT isoform-selective. Docking studies suggest that only one flavonoid can bind to GLUT1 at any instant, but sugar transport and ligand-binding studies indicate that human erythrocyte GLUT1 can bind at least two flavonoid molecules simultaneously. Quercetin and EGCG are each characterized by positive, cooperative binding, whereas ECG shows negative cooperative binding. These findings support recent studies suggesting that GLUT1 forms an oligomeric complex of interacting, allosteric, alternating access transporters. We discuss how modulation of facilitative glucose transporters could contribute to the protective actions of the flavonoids against diabetes and Alzheimer's disease.

Do Skeletal Dynamics Mediate Sugar Uptake and Transport in Human Erythrocytes?

We explore, herein, the hypothesis that transport of molecules or ions into erythrocytes may be affected and directly stimulated by the dynamics of the spectrin/actin skeleton. Skeleton/actin motions are driven by thermal fluctuations that may be influenced by ATP hydrolysis as well as by structural alterations of the junctional complexes that connect the skeleton to the cell's lipid membrane. Specifically, we focus on the uptake of glucose into erythrocytes via glucose transporter 1 and on the kinetics of glucose disassociation at the endofacial side of glucose transporter 1. We argue that glucose disassociation is affected by both hydrodynamic forces induced by the actin/spectrin skeleton and by probable contact of the swinging 37-nm-long F-actin protofilament with glucose, an effect we dub the "stickball effect." Our hypothesis and results are interpreted within the framework of the kinetic measurements and compartmental kinetic models of Carruthers and co-workers; these experimental results and models describe glucose disassociation as the "slow step" (i.e., rate-limiting step) in the uptake process. Our hypothesis is further supported by direct simulations of skeleton-enhanced transport using our molecular-based models for the actin/spectrin skeleton as well as by experimental measurements of glucose uptake into cells subject to shear deformations, which demonstrate the hydrodynamic effects of advection. Our simulations have, in fact, previously demonstrated enhanced skeletal dynamics in cells in shear deformations, as they occur naturally within the skeleton, which is an effect also supported by experimental observations.

Kinetic Basis of Cis- and Trans-Allostery in GLUT1-Mediated Sugar Transport.

A growing body of evidence demonstrates that GLUT1-mediated erythrocyte sugar transport is more complex than widely assumed and that contemporary interpretations of emergent GLUT1 structural data are incompatible with the available transport and biochemical data. This study examines the kinetic basis of one such incompatibility-transport allostery-and in doing so suggests how the results of studies examining GLUT1 structure and function may be reconciled. Three types of allostery are observed in GLUT1-mediated, human erythrocyte sugar transport: (1) exofacial cis-allostery in which low concentrations of extracellular inhibitors stimulate sugar uptake while high concentrations inhibit transport; (2) endofacial cis-allostery in which low concentrations of intracellular inhibitors enhance cytochalasin B binding to GLUT1 while high concentrations inhibit binding, and (3) trans-allostery in which low concentrations of ligands acting at one cell surface stimulate ligand binding at or sugar transport from the other surface while high concentrations inhibit these processes. We consider several kinetic models to account for these phenomena. Our results show that an inhibitor can only stimulate then inhibit sugar uptake if (1) the transporter binds two or more molecules of inhibitor; (2) high-affinity binding to the first site stimulates transport, and (3) low-affinity binding to the second site inhibits transport. Reviewing the available structural, transport, and ligand binding data, we propose that exofacial cis-allostery results from cross-talk between multiple, co-existent ligand interaction sites present in the exofacial cavity of each GLUT1 protein, whereas trans-allostery and endofacial cis-allostery require ligand-induced subunit-subunit interactions.

Reconciling contradictory findings: Glucose transporter 1 (GLUT1) functions as an oligomer of allosteric, alternating access transporters.

Recent structural studies suggest that GLUT1 (glucose transporter 1)-mediated sugar transport is mediated by an alternating access transporter that successively presents exofacial (e2) and endofacial (e1) substrate-binding sites. Transport studies, however, indicate multiple, interacting (allosteric), and co-existent, exo- and endofacial GLUT1 ligand-binding sites. The present study asks whether these contradictory conclusions result from systematic analytical error or reveal a more fundamental relationship between transporter structure and function. Here, homology modeling supported the alternating access transporter model for sugar transport by confirming at least four GLUT1 conformations, the so-called outward, outward-occluded, inward-occluded, and inward GLUT1 conformations. Results from docking analysis suggested that outward and outward-occluded conformations present multiple ß-d-glucose and maltose interaction sites, whereas inward-occluded and inward conformations present only a single ß-d-glucose interaction site. Gln-282 contributed to sugar binding in all GLUT1 conformations via hydrogen bonding. Mutating Gln-282 to alanine (Q282A) doubled the Km(app) for 2-deoxy-d-glucose uptake and eliminated cis-allostery (stimulation of sugar uptake by subsaturating extracellular maltose) but not trans-allostery (uptake stimulation by subsaturating cytochalasin B). cis-Allostery persisted, but trans-allostery was lost in an oligomerization-deficient GLUT1 variant in which we substituted membrane helix 9 with the equivalent GLUT3 sequence. Moreover, Q282A eliminated cis-allostery in the oligomerization variant. These findings reconcile contradictory conclusions from structural and transport studies by suggesting that GLUT1 is an oligomer of allosteric, alternating access transporters in which 1) cis-allostery is mediated by intrasubunit interactions and 2) trans-allostery requires intersubunit interactions.

WZB117 (2-Fluoro-6-(m-hydroxybenzoyloxy) Phenyl m-Hydroxybenzoate) Inhibits GLUT1-mediated Sugar Transport by Binding Reversibly at the Exofacial Sugar Binding Site.

WZB117 (2-fluoro-6-(m-hydroxybenzoyloxy) phenyl m-hydroxybenzoate) inhibits passive sugar transport in human erythrocytes and cancer cell lines and, by limiting glycolysis, inhibits tumor growth in mice. This study explores how WZB117 inhibits the erythrocyte sugar transporter glucose transport protein 1 (GLUT1) and examines the transporter isoform specificity of inhibition. WZB117 reversibly and competitively inhibits erythrocyte 3-O-methylglucose (3MG) uptake with Ki(app) = 6 µm but is a noncompetitive inhibitor of sugar exit. Cytochalasin B (CB) is a reversible, noncompetitive inhibitor of 3MG uptake with Ki(app) = 0.3 µm but is a competitive inhibitor of sugar exit indicating that WZB117 and CB bind at exofacial and endofacial sugar binding sites, respectively. WZB117 inhibition of GLUTs expressed in HEK293 cells follows the order of potency: insulin-regulated GLUT4 ≫ GLUT1 ≈ neuronal GLUT3. This may explain WZB117-induced murine lipodystrophy. Molecular docking suggests the following. 1) The WZB117 binding envelopes of exofacial GLUT1 and GLUT4 conformers differ significantly. 2) GLUT1 and GLUT4 exofacial conformers present multiple, adjacent glucose binding sites that overlap with WZB117 binding envelopes. 3) The GLUT1 exofacial conformer lacks a CB binding site. 4) The inward GLUT1 conformer presents overlapping endofacial WZB117, d-glucose, and CB binding envelopes. Interrogating the GLUT1 mechanism using WZB117 reveals that subsaturating WZB117 and CB stimulate erythrocyte 3MG uptake. Extracellular WZB117 does not affect CB binding to GLUT1, but intracellular WZB117 inhibits CB binding. These findings are incompatible with the alternating conformer carrier for glucose transport but are consistent with either a multisubunit, allosteric transporter, or a transporter in which each subunit presents multiple, interacting ligand binding sites.

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

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.

A novel model for brain iron uptake: introducing the concept of regulation.

Neurologic disorders such as Alzheimer's, Parkinson's disease, and Restless Legs Syndrome involve a loss of brain iron homeostasis. Moreover, iron deficiency is the most prevalent nutritional concern worldwide with many associated cognitive and neural ramifications. Therefore, understanding the mechanisms by which iron enters the brain and how those processes are regulated addresses significant global health issues. The existing paradigm assumes that the endothelial cells (ECs) forming the blood-brain barrier (BBB) serve as a simple conduit for transport of transferrin-bound iron. This concept is a significant oversimplification, at minimum failing to account for the iron needs of the ECs. Using an in vivo model of brain iron deficiency, the Belgrade rat, we show the distribution of transferrin receptors in brain microvasculature is altered in luminal, intracellular, and abluminal membranes dependent on brain iron status. We used a cell culture model of the BBB to show the presence of factors that influence iron release in non-human primate cerebrospinal fluid and conditioned media from astrocytes; specifically apo-transferrin and hepcidin were found to increase and decrease iron release, respectively. These data have been integrated into an interactive model where BBB ECs are central in the regulation of cerebral iron metabolism.

Human erythrocytes transport dehydroascorbic acid and sugars using the same transporter complex.

GLUT1, the primary glucose transport protein in human erythrocytes [red blood cells (RBCs)], also transports oxidized vitamin C [dehydroascorbic acid (DHA)]. A recent study suggests that RBC GLUT1 transports DHA as its primary substrate and that only a subpopulation of GLUT1 transports sugars. This conclusion is based on measurements of cellular glucose and DHA equilibrium spaces, rather than steady-state transport rates. We have characterized RBC transport of DHA and 3-O-methylglucose (3-OMG), a transported, nonmetabolizable sugar. Steady-state 3-OMG and DHA uptake in the absence of intracellular substrate are characterized by similar Vmax (0.16 ± 0.01 and 0.13 ± 0.02 mmol·l(-1)·min(-1), respectively) and apparent Km (1.4 ± 0.2 and 1.6 ± 0.7 mM, respectively). 3-OMG and DHA compete for uptake, with Ki(app) of 0.7 ± 0.4 and 1.1 ± 0.1 mM, respectively. Uptake measurements using RBC inside-out-membrane vesicles demonstrate that 3-OMG and DHA compete at the cytoplasmic surface of the membrane, with Ki(app) of 0.7 ± 0.1 and 0.6 ± 0.1 mM, respectively. Intracellular 3-OMG stimulates unidirectional uptake of 3-OMG and DHA. These findings indicate that DHA and 3-OMG bind at mutually exclusive sites at exo- and endofacial surfaces of GLUT1 and are transported via the same GLUT1 complex.

Sequence determinants of GLUT1 oligomerization: analysis by homology-scanning mutagenesis.

The human blood-brain barrier glucose transport protein (GLUT1) forms homodimers and homotetramers in detergent micelles and in cell membranes, where the GLUT1 oligomeric state determines GLUT1 transport behavior. GLUT1 and the neuronal glucose transporter GLUT3 do not form heterocomplexes in human embryonic kidney 293 (HEK293) cells as judged by co-immunoprecipitation assays. Using homology-scanning mutagenesis in which GLUT1 domains are substituted with equivalent GLUT3 domains and vice versa, we show that GLUT1 transmembrane helix 9 (TM9) is necessary for optimal association of GLUT1-GLUT3 chimeras with parental GLUT1 in HEK cells. GLUT1 TMs 2, 5, 8, and 11 also contribute to a less abundant heterocomplex. Cell surface GLUT1 and GLUT3 containing GLUT1 TM9 are 4-fold more catalytically active than GLUT3 and GLUT1 containing GLUT3 TM9. GLUT1 and GLUT3 display allosteric transport behavior. Size exclusion chromatography of detergent solubilized, purified GLUT1 resolves GLUT1/lipid/detergent micelles as 6- and 10-nm Stokes radius particles, which correspond to GLUT1 dimers and tetramers, respectively. Studies with GLUTs expressed in and solubilized from HEK cells show that HEK cell GLUT1 resolves as 6- and 10-nm Stokes radius particles, whereas GLUT3 resolves as a 6-nm particle. Substitution of GLUT3 TM9 with GLUT1 TM9 causes chimeric GLUT3 to resolve as 6- and 10-nm Stokes radius particles. Substitution of GLUT1 TM9 with GLUT3 TM9 causes chimeric GLUT1 to resolve as a mixture of 6- and 4-nm particles. We discuss these findings in the context of determinants of GLUT oligomeric structure and transport function.

Sequence determinants of GLUT1-mediated accelerated-exchange transport: analysis by homology-scanning mutagenesis.

The class 1 equilibrative glucose transporters GLUT1 and GLUT4 are structurally similar but catalyze distinct modes of transport. GLUT1 exhibits trans-acceleration, in which the presence of intracellular sugar stimulates the rate of unidirectional sugar uptake. GLUT4-mediated uptake is unaffected by intracellular sugar. Using homology-scanning mutagenesis in which domains of GLUT1 are substituted with equivalent domains from GLUT4 and vice versa, we show that GLUT1 transmembrane domain 6 is both necessary and sufficient for trans-acceleration. This region is not directly involved in GLUT1 binding of substrate or inhibitors. Rather, transmembrane domain 6 is part of two putative scaffold domains, which coordinate membrane-spanning amphipathic helices that form the sugar translocation pore. We propose that GLUT1 transmembrane domain 6 restrains import when intracellular sugar is absent by slowing transport-associated conformational changes.

Role of monosaccharide transport proteins in carbohydrate assimilation, distribution, metabolism, and homeostasis.

The facilitated diffusion of glucose, galactose, fructose, urate, myoinositol, and dehydroascorbicacid in mammals is catalyzed by a family of 14 monosaccharide transport proteins called GLUTs. These transporters may be divided into three classes according to sequence similarity and function/substrate specificity. GLUT1 appears to be highly expressed in glycolytically active cells and has been coopted in vitamin C auxotrophs to maintain the redox state of the blood through transport of dehydroascorbate. Several GLUTs are definitive glucose/galactose transporters, GLUT2 and GLUT5 are physiologically important fructose transporters, GLUT9 appears to be a urate transporter while GLUT13 is a proton/myoinositol cotransporter. The physiologic substrates of some GLUTs remain to be established. The GLUTs are expressed in a tissue specific manner where affinity, specificity, and capacity for substrate transport are paramount for tissue function. Although great strides have been made in characterizing GLUT-catalyzed monosaccharide transport and mapping GLUT membrane topography and determinants of substrate specificity, a unifying model for GLUT structure and function remains elusive. The GLUTs play a major role in carbohydrate homeostasis and the redistribution of sugar-derived carbons among the various organ systems. This is accomplished through a multiplicity of GLUT-dependent glucose sensing and effector mechanisms that regulate monosaccharide ingestion, absorption,distribution, cellular transport and metabolism, and recovery/retention. Glucose transport and metabolism have coevolved in mammals to support cerebral glucose utilization.

AMP kinase regulation of sugar transport in brain capillary endothelial cells during acute metabolic stress.

AMP-dependent kinase (AMPK) and GLUT1-mediated sugar transport in blood-brain barrier endothelial cells are activated during acute cellular metabolic stress. Using murine brain microvasculature endothelium bEnd.3 cells, we show that AMPK phosphorylation and stimulation of 3-O-methylglucose transport by the AMPK agonist AICAR are inhibited in a dose-dependent manner by the AMPK antagonist Compound C. AMPK α1- or AMPK α2-knockdown by RNA interference or AMPK inhibition by Compound C reduces AMPK phosphorylation and 3-O-methylglucose transport stimulation induced by cellular glucose-depletion, by potassium cyanide (KCN), or by carbonyl cyanide-p-trifluoromethoxy-phenylhydrazone (FCCP). Cell surface biotinylation studies reveal that plasma membrane GLUT1 levels are increased two- to threefold by cellular glucose depletion, AICAR or KCN treatment, and that these increases are prevented by Compound C and by AMPK α1- or α2-knockdown. These results support the hypothesis that AMPK activation in blood-brain barrier-derived endothelial cells directs the trafficking of GLUT1 intracellular pools to the plasma membrane, thereby increasing endothelial sugar transport capacity.

Response to 'comment on recent modeling studies of astrocyte-neuron metabolic interactions': much ado about nothing.

For many years, a tenet of cerebral metabolism held that glucose was the obligate energy substrate of the mammalian brain and that neuronal oxidative metabolism represented the majority of this glucose utilization. In 1994, Pellerin and Magistretti formulated the astrocyte-neuron lactate shuttle (ANLS) hypothesis, in which astrocytes, not neurons, metabolized glucose, with subsequent transport of the glycolytically derived lactate to fuel the energy needs of the neuron during neurotransmission. By considering the concentrations and kinetic characteristics of the nutrient transporter proteins, Simpson et al later supported the opposite view, in which lactate flows from neurons to astrocytes, thus leading to the neuron-astrocyte lactate shuttle (NALS). Most recently, a commentary was published in this journal attempting to discredit the NALS. This challenge has stimulated the present response in which we detail the inaccuracies of the commentary and further model several different possibilities. Although our simulations continue to support the predominance of neuronal glucose utilization during activation and neuronal to astrocytic lactate flow, the most important result is that, regardless of the direction of the flow, the overall contribution of lactate to cerebral glucose metabolism is found to be so small as to make this ongoing debate 'much ado about nothing'.

Determinants of ligand binding affinity and cooperativity at the GLUT1 endofacial site.

Cytochalasin B (CB) and forskolin (FSK) inhibit GLUT1-mediated sugar transport in red cells by binding at or close to the GLUT1 endofacial sugar binding site. Paradoxically, very low concentrations of each of these inhibitors produce a modest stimulation of sugar transport [ Cloherty, E. K., Levine, K. B., and Carruthers, A. ((2001)) The red blood cell glucose transporter presents multiple, nucleotide-sensitive sugar exit sites. Biochemistry 40 ((51)) 15549-15561]. This result is consistent with the hypothesis that the glucose transporter contains multiple, interacting, endofacial binding sites for CB and FSK. The present study tests this hypothesis directly and, by screening a library of cytochalasin and forskolin analogues, asks what structural features of endofacial site ligands determine binding site affinity and cooperativity. Like CB, FSK competitively inhibits exchange 3-O-methylglucose transport (sugar uptake in cells containing intracellular sugar) but noncompetitively inhibits sugar uptake into cells lacking sugar at 4 °C. This refutes the hypothesis that FSK binds at GLUT1 endofacial and exofacial sugar binding sites. Some forskolin derivatives and cytochalasins inhibit equilibrium [(3)H]-CB binding to red cell membranes depleted of peripheral proteins at 4 °C. Others produce a moderate stimulation of [(3)H]-CB binding when introduced at low concentrations but inhibit binding as their concentration is increased. Yet other analogues modestly stimulate [(3)H]-CB binding at all inhibitor concentrations applied. These findings are explained by a carrier that presents at least two interacting endofacial binding sites for CB or FSK. We discuss this result within the context of models for GLUT1-mediated sugar transport and GLUT1 quaternary structure, and we evaluate the major determinants of ligand binding affinity and cooperativity.

Acute modulation of sugar transport in brain capillary endothelial cell cultures during activation of the metabolic stress pathway.

GLUT1-catalyzed equilibrative sugar transport across the mammalian blood-brain barrier is stimulated during acute and chronic metabolic stress; however, the mechanism of acute transport regulation is unknown. We have examined acute sugar transport regulation in the murine brain microvasculature endothelial cell line bEnd.3. Acute cellular metabolic stress was induced by glucose depletion, by potassium cyanide, or by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, which reduce or deplete intracellular ATP within 15 min. This results in a 1.7-7-fold increase in V(max) for zero-trans 3-O-methylglucose uptake (sugar uptake into sugar-free cells) and a 3-10-fold increase in V(max) for equilibrium exchange transport (intracellular [sugar] = extracellular [sugar]). GLUT1, GLUT8, and GLUT9 mRNAs are detected in bEnd.3 cells where GLUT1 mRNA levels are 33-fold greater than levels of GLUT8 or GLUT9 mRNA. Neither GLUT1 mRNA nor total protein levels are affected by acute metabolic stress. Cell surface biotinylation reveals that plasma membrane GLUT1 levels are increased 2-3-fold by metabolic depletion, although cell surface Na(+),K(+)-ATPase levels remain unaffected by ATP depletion. Treatment with the AMP-activated kinase agonist, AICAR, increases V(max) for net 3-O-methylglucose uptake by 2-fold. Glucose depletion and treatment with potassium cyanide, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and AICAR also increase AMP-dependent kinase phosphorylation in bEnd.3 cells. These results suggest that metabolic stress rapidly stimulates blood-brain barrier endothelial cell sugar transport by acute up-regulation of plasma membrane GLUT1 levels, possibly involving AMP-activated kinase activity.

Will the original glucose transporter isoform please stand up!

Monosaccharides enter cells by slow translipid bilayer diffusion by rapid, protein-mediated, cation-dependent cotransport and by rapid, protein-mediated equilibrative transport. This review addresses protein-mediated, equilibrative glucose transport catalyzed by GLUT1, the first equilibrative glucose transporter to be identified, purified, and cloned. GLUT1 is a polytopic, membrane-spanning protein that is one of 13 members of the human equilibrative glucose transport protein family. We review GLUT1 catalytic and ligand-binding properties and interpret these behaviors in the context of several putative mechanisms for protein-mediated transport. We conclude that no single model satisfactorily explains GLUT1 behavior. We then review GLUT1 topology, subunit architecture, and oligomeric structure and examine a new model for sugar transport that combines structural and kinetic analyses to satisfactorily reproduce GLUT1 behavior in human erythrocytes. We next review GLUT1 cell biology and the transcriptional and posttranscriptional regulation of GLUT1 expression in the context of development and in response to glucose perturbations and hypoxia in blood-tissue barriers. Emphasis is placed on transgenic GLUT1 overexpression and null mutant model systems, the latter serving as surrogates for the human GLUT1 deficiency syndrome. Finally, we review the role of GLUT1 in the absence or deficiency of a related isoform, GLUT3, toward establishing the physiological significance of coordination between these two isoforms.

The in vivo neuron-to-astrocyte lactate shuttle in human brain: evidence from modeling of measured lactate levels during visual stimulation.

Functional magnetic resonance spectroscopy (fMRS) allows the non-invasive measurement of metabolite concentrations in the human brain, including changes induced by variations in neurotransmission activity. However, the limited spatial and temporal resolution of fMRS does not allow specific measurements of metabolites in different cell types. Thus, the analysis of fMRS data in the context of compartmentalized metabolism requires the formulation and application of mathematical models. In the present study we utilized the mathematical model introduced by Simpson et al. (2007) to gain insights into compartmentalized metabolism in vivo from the fMRS data obtained in humans at ultra high magnetic field by Mangia et al. (2007a). This model simulates brain glucose and lactate levels in a theoretical cortical slice. Using experimentally determined concentrations and catalytic activities for the respective transporter proteins, we calculate inflow and export of glucose and lactate in endothelium, astrocytes, and neurons. We then vary neuronal and astrocytic glucose and lactate utilization capacities until close correspondence is observed between in vivo and simulated glucose and lactate levels. The results of the simulations indicate that, when literature values of glucose transport capacity are utilized, the fMRS data are consistent with export of lactate by neurons and import of lactate by astrocytes, a mechanism that can be referred to as a neuron-to-astrocyte lactate shuttle. A shuttle of lactate from astrocytes to neurons could be simulated, but this required the astrocytic glucose transport capacity to be increased by 12-fold, and required that neurons not respond to activation with increased glycolysis, two conditions that are not supported by current literature.

alpha- and beta-monosaccharide transport in human erythrocytes.

Equilibrative sugar uptake in human erythrocytes is characterized by a rapid phase, which equilibrates 66% of the cell water, and by a slow phase, which equilibrates 33% of the cell water. This behavior has been attributed to the preferential transport of beta-sugars by erythrocytes (Leitch JM, Carruthers A. Am J Physiol Cell Physiol 292: C974-C986, 2007). The present study tests this hypothesis. The anomer theory requires that the relative compartment sizes of rapid and slow transport phases are determined by the proportions of beta- and alpha-sugar in aqueous solution. This is observed with D-glucose and 3-O-methylglucose but not with 2-deoxy-D-glucose and D-mannose. The anomer hypothesis predicts that the slow transport phase, which represents alpha-sugar transport, is eliminated when anomerization is accelerated to generate the more rapidly transported beta-sugar. Exogenous, intracellular mutarotase accelerates anomerization but has no effect on transport. The anomer hypothesis requires that transport inhibitors inhibit rapid and slow transport phases equally. This is observed with the endofacial site inhibitor cytochalasin B but not with the exofacial site inhibitors maltose or phloretin, which inhibit only the rapid phase. Direct measurement of alpha- and beta-sugar uptake demonstrates that erythrocytes transport alpha- and beta-sugars with equal avidity. These findings refute the hypothesis that erythrocytes preferentially transport beta-sugars. We demonstrate that biphasic 3-O-methylglucose equilibrium exchange kinetics refute the simple carrier hypothesis for protein-mediated sugar transport but are compatible with a fixed-site transport mechanism regulated by intracellular ATP and cell shape.

Analysis of glucose transporter topology and structural dynamics.

Homology modeling and scanning cysteine mutagenesis studies suggest that the human glucose transport protein GLUT1 and its distant bacterial homologs LacY and GlpT share similar structures. We tested this hypothesis by mapping the accessibility of purified, reconstituted human erythrocyte GLUT1 to aqueous probes. GLUT1 contains 35 potential tryptic cleavage sites. Fourteen of 16 lysine residues and 18 of 19 arginine residues were accessible to trypsin. GLUT1 lysine residues were modified by isothiocyanates and N-hydroxysuccinimide (NHS) esters in a substrate-dependent manner. Twelve lysine residues were accessible to sulfo-NHS-LC-biotin. GLUT1 trypsinization released full-length transmembrane helix 1, cytoplasmic loop 6-7, and the long cytoplasmic C terminus from membranes. Trypsin-digested GLUT1 retained cytochalasin B and d-glucose binding capacity and released full-length transmembrane helix 8 upon cytochalasin B (but not D-glucose) binding. Transmembrane helix 8 release did not abrogate cytochalasin B binding. GLUT1 was extensively proteolyzed by alpha-chymotrypsin, which cuts putative pore-forming amphipathic alpha-helices 1, 2, 4, 7, 8, 10, and 11 at multiple sites to release transmembrane peptide fragments into the aqueous solvent. Putative scaffolding membrane helices 3, 6, 9, and 12 are strongly hydrophobic, resistant to alpha-chymotrypsin, and retained by the membrane bilayer. These observations provide experimental support for the proposed GLUT1 architecture; indicate that the proposed topology of membrane helices 5, 6, and 12 requires adjustment; and suggest that the metastable conformations of transmembrane helices 1 and 8 within the GLUT1 scaffold destabilize a sugar translocation intermediate.