DOCSLIB.ORG

Crystal Structure of a Glucose/H Symporter and Its Mechanism of Action

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Crystal Structure of a Glucose/H Symporter and Its Mechanism of Action + Crystal structure of a glucose/H symporter and its mechanism of action Cristina V. Iancua, Jamillah Zamoonb, Sang Bum Wooa, Alexander Aleshinc, and Jun-yong Choea,1 aDepartment of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science, The Chicago Medical School, North Chicago,IL 60064; bDepartment of Biological Sciences, Faculty of Science, Kuwait University, Kuwait City 13060, Kuwait; and cDepartment of Infectious Diseases, Sanford Burnham Medical Research Institute, La Jolla, CA 92037 Edited* by H. Ronald Kaback, University of California, Los Angeles, CA, and approved September 26, 2013 (received for review June 25, 2013) + Glucose transporters are required to bring glucose into cells, of a Staphylococcus epidermidis glucose/H symporter (GlcPSe) where it is an essential energy source and precursor in protein by single anomalous dispersion methods. GlcPSe shares high se- and lipid synthesis. These transporters are involved in important quence identity (27–34%) and homology (49–58%) with the hu- common diseases such as cancer and diabetes. Here, we report the man GLUTs (Table S1), is highly specific for glucose, and is + crystal structure of the Staphylococcus epidermidis glucose/H sym- inhibited by the well-characterized inhibitors of human GLUTs porter in an inward-facing conformation at 3.2-Å resolution. The phloretin, cytochalasin B, and forskolin. In contrast to GlcP , + Se Staphylococcus epidermidis glucose/H symporter is homologous XylE transports xylose but not glucose, which is an inhibitor, and is to human glucose transporters, is very specific and has high avidity impervious to inhibition by cytochalasin B (13, 24). On the basis of for glucose, and is inhibited by the human glucose transport inhib- the GlcPSe structure and functional studies of wild-type and mu- itors cytochalasin B, phloretin, and forskolin. On the basis of the tant transporters, we identify residues from helices 1 and 4 that are crystal structure in conjunction with mutagenesis and functional + + important for glucose/H symport (most bacterial glucose trans- studies, we propose a mechanism for glucose/H symport and dis- porters) versus uniport (most mammalian GLUTs) and propose + cuss the symport mechanism versus facilitated diffusion. a kinetic mechanism for glucose/H symport. major facilitator superfamily | membrane protein | GLUT | Results sugar transporter | solute-carrier 2A With the goal of determining the 3D structure of a glucose BIOCHEMISTRY transporter, we screened more than 50 human, archaeal, and lucose and related sugars are vital to most living cells as bacterial genes and proteins based on BLAST searches of human Ga source of both energy and carbon. In animal cells, facili- GLUTs and their homologs for protein overexpression, purifi- tated diffusion of glucose and related monosaccharides occurs cation, transport activity, crystallization, and X-ray diffraction. through members of the solute-carrier 2A or glucose transport S. epidermidis had a high score in BLAST searches against many (GLUT) family. In humans, 14 members of the GLUT family human GLUTs (Table S2). Because the gene (ZP-12295482.1) have been identified. Despite significant sequence homology product exhibited significant specific activity for glucose transport, – (19 65% identity) (Table S1), they differ in tissue distribution, we named it “GlcPSe.” Diffracting crystals (up to 5 Å) were ob- level of expression, substrate specificity, and affinity, presumably tained for several different bacterial gene products. Among all the to suit the physiological needs of a particular tissue (1, 2). transporters that yielded diffracting crystals, GlcPSe had the most GLUTs have been implicated in GLUT deficiency syndrome (3), robust glucose transporter activity. Fanconi-Bickel syndrome (4), cancer (5, 6) and diabetes (7). + + Except for GLUT13, which is a myo-inositol/H symporter (8), GlcPSe Is a Glucose/H Symporter. We tested GlcPSe for glucose + and GLUT12, which functions as a glucose/H symporter (9), all transport using whole cells, right-side-out (RSO) vesicles, and others presumably are uniporters that catalyze equilibration of proteoliposomes (Fig. 1). Glucose transport was linear for at glucose across the membrane. In addition to human GLUTs, glucose transporters are important for understanding glucose Significance uptake and metabolism during sugar fermentation and alcohol production via a myriad of hexose transporters in yeasts (10). In Glucose transporters mediate the exchange of glucose and re- plants, monosaccharide and sucrose transporters play a funda- lated hexoses in living cells. In humans, these transporters mental role in stress responses and developmental processes (known as GLUT) are involved in several diseases, including including seed germination and balanced growth (11, 12). cancer and diabetes. The glucose transporter from Staphylo- GLUTs belong to the major facilitator superfamily (MFS), coccus epidermidis (GlcPSe) has high sequence homology to one of the largest protein families containing more than 10,000 human GLUT, is specific for glucose, and is inhibited by human members (www.tcdb.org). However, 3D structures are available GLUT inhibitors. The crystal structure of GlcPSe, along with site- for only nine MFS proteins (13–21), and none is a true glucose directed mutagenesis and transport-activity studies, provide transporter. The most accepted model for transport by MFS insight into the mechanism of glucose transport. proteins is alternating access in which the substrate-binding site is alternatively exposed to either side of the membrane (22). The Author contributions: J.-y.C. designed research; C.V.I., J.Z., S.B.W., A.A., and J.-y.C. per- available MFS X-ray crystal structures are consistent with such formed research; C.V.I., J.Z., S.B.W., A.A., and J.-y.C. analyzed data; and C.V.I. and J.-y.C. wrote the paper. a model and represent inward-facing (open to the cytoplasmic The authors declare no conflict of interest. side) [as in LacY (15), GlpT (16), PepTSo (19), and NarK (20)], outward-facing (open to the periplasmic side) [as in FucP (18) *This Direct Submission article had a prearranged editor. and XylE (13)], and occluded conformations [as in EmrD (17), Data deposition: Crystallography, atomic coordinates, and structure factors reported in this paper have been deposited in the Protein Data Bank (PDB), www.pdb.org (PDB ID OxlT (14), and NarU (21)]. code 4LDS). Recently the crystal structures of XylE, a glucose transporter 1To whom correspondence should be addressed. E-mail: junyong.choe@rosalindfranklin. homolog from Escherichia coli, in outward-facing, substrate-bound edu. conformation (13), and inward-facing conformation (23) were de- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. termined. Here we report the inward-facing, unliganded structure 1073/pnas.1311485110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1311485110 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 counterflow activity for glucose with an optimum at acidic pH (Fig. 1E). Effect of Human GLUT Inhibitors on the Glucose Transport of GlcPSe. Previous work with human GLUTs identified several inhibitors of glucose transport: cytochalasin B, phloretin, forskolin, and phlorizin (28, 29). Among these, phloretin fully inhibited the transport activity of GlcPSe in whole cells and RSO vesicles (Fig. 2). Cytochalasin B and forskolin partially inhibited GlcPSe (75%) in RSO vesicles. Partial inhibition by cytochalasin B has been reported for the glucose transporter from Trypanosoma brucei (30). The phloretin IC50 was 153 ± 25 μM(Fig.2B). The relative IC50 for cytochalasin B and forskolin was 18.1 ± 6.3 μM, and 2.35 ± 0.41 μM, respectively (Fig. 2B). Phlorizin did not inhibit the glucose transport significantly at the concentrations used (up to 1 mM). Inward-Facing Conformation. The structure of GlcPSe (Table S3 and Fig. S1) exhibits the trademark topology of MFS proteins: 12 transmembrane helices organized into two six-helix bundles, with the N and C domains related by pseudo two-fold symmetry (Fig. 3). The N and C domains can be superimposed with an rmsd of Fig. 1. Glucose transport activity of GlcPSe.(A) Glucose uptake was mea- 14 sured with 30 μMof C-glucose, in RSO vesicles with GlcPSe (GlcPSe)or 2.8 Å. They are connected by a long cytoplasmic loop located without GlcPSe (control). After 15 min glucose uptake reached steady state. between transmembrane (TM) helices 6 and 7, and the N and C + To determine the ratio of H :glucose symport, 10 mM of unlabeled glucose termini are on the cytoplasmic face of the membrane. Each – was added after 20 min. (B) Michaelis Menten plot of GlcPSe glucose trans- corresponding domain from the outward-facing conformation of port. Transport activity was measured at a total glucose concentration of 5– XylE (13) and the inward-facing conformation of GlcP can be μ Se 750 M. Error bars indicate SD from nine data points of both RSO vesicles superimposed with an rmsd of 1.7 Å for the N domains and 2.3 Å ± μ ± · −1· −1 and intact cells. Km (29 4 M) and Vmax (160 6 nmol min mg protein ) for the C domains. Therefore, the transition from the inward- to were determined by nonlinear regression enzyme kinetics. The y-axis was normalized based on GlcP concentration as determined from Western the outward-facing conformations appears to involve general rigid Se body movements of the two halves (Movie S1). The outward- blotting. (C) Glucose uptake in RSO vesicles for GlcPSe (Wt) in various con- + + ditions. The electrochemical H gradient, Δμ̃H , was generated by adding 0.2 facing conformation of GlcPSe was modeled on the basis of XylE mM phenazine methosulfate (PMS) and 20 mM Ascorbate (Asc). The trans- [Protein Data Bank (PDB) ID code 4GC0] (13), and the inward- μ 14 port reaction was initiated by the addition of 25 M C-glucose. The effects and outward-facing conformations of GlcPSe were compared. On of pH (5.5 or 7.5) and the protonophore CCCP (10 μM) on glucose uptake the cytoplasmic side, the TM helices of GlcPSe are moved further were examined.
Load more

Recommended publications
  • Screening and Identification of Key Biomarkers in Clear Cell Renal Cell Carcinoma Based on Bioinformatics Analysis
    bioRxiv preprint doi: https://doi.org/10.1101/2020.12.21.423889; this version posted December 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Screening and identification of key biomarkers in clear cell renal cell carcinoma based on bioinformatics analysis Basavaraj Vastrad1, Chanabasayya Vastrad*2 , Iranna Kotturshetti 1. Department of Biochemistry, Basaveshwar College of Pharmacy, Gadag, Karnataka 582103, India. 2. Biostatistics and Bioinformatics, Chanabasava Nilaya, Bharthinagar, Dharwad 580001, Karanataka, India. 3. Department of Ayurveda, Rajiv Gandhi Education Society`s Ayurvedic Medical College, Ron, Karnataka 562209, India. * Chanabasayya Vastrad [email protected] Ph: +919480073398 Chanabasava Nilaya, Bharthinagar, Dharwad 580001 , Karanataka, India bioRxiv preprint doi: https://doi.org/10.1101/2020.12.21.423889; this version posted December 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Clear cell renal cell carcinoma (ccRCC) is one of the most common types of malignancy of the urinary system. The pathogenesis and effective diagnosis of ccRCC have become popular topics for research in the previous decade. In the current study, an integrated bioinformatics analysis was performed to identify core genes associated in ccRCC. An expression dataset (GSE105261) was downloaded from the Gene Expression Omnibus database, and included 26 ccRCC and 9 normal kideny samples. Assessment of the microarray dataset led to the recognition of differentially expressed genes (DEGs), which was subsequently used for pathway and gene ontology (GO) enrichment analysis.
    [Show full text]
  • (Glut1ds): Methylxanthines Potentiate GLUT1 Haploinsufficiency in Vitro
    0031-3998/01/5002-0254 PEDIATRIC RESEARCH Vol. 50, No. 2, 2001 Copyright © 2001 International Pediatric Research Foundation, Inc. Printed in U.S.A. Glucose Transporter Type 1 Deficiency Syndrome (Glut1DS): Methylxanthines Potentiate GLUT1 Haploinsufficiency In Vitro YUAN-YUAN HO, HONG YANG, JÖRG KLEPPER, JORGE FISCHBARG, DONG WANG, AND DARRYL C. DE VIVO Department of Neurology, Columbia University, New York, New York 10032, U.S.A. [Y.Y.H., H.Y., D.W., D.C.D.]; Department of Pediatrics, University of Essen, Essen, Germany 45122 [J.K.]; and Departments of Physiology and Cellular Biophysics, and Ophthalmology, Columbia University, New York, New York 10032, U.S.A. [J.F.] ABSTRACT Methylxanthines such as caffeine and theophylline are known substrate for Glut1. The combined effects of caffeine (3 mM) and to inhibit glucose transport. We have studied such inhibition in phenobarbital (10 mM) on glucose transport, as determined in the glucose transporter type 1 deficiency syndrome (Glut1DS) by patient 15 and the maternal control, show no additive or syner- erythrocyte glucose transport assays. Data from four patients gistic inhibition. These data indicate that caffeine and phenobar- with individual mutations in the GLUT1 gene are discussed: bital have similar Glut1 inhibitory properties in these two sub- patient 1 (hemizygosity), 3 (S66F), 15 (368Ins23), and 17 jects. Our study suggests that Glut1DS patients may have a (R333W). Zero-trans influx of 14C-labeled 3-O-methyl glucose reduced safety margin for methylxanthines. Consumption of (3-OMG) into erythrocytes of patients is reduced (patient 1, 51%; methylxanthine-containing products may aggravate the neuro- 3, 45%; 15, 31%; 17, 52%) compared with maternal controls.
    [Show full text]
  • Regulation of Myocardial Glucose Transporters GLUT1 and GLUT4 in Chronically Anemic Fetal Lambs
    0031-3998/05/5804-0713 PEDIATRIC RESEARCH Vol. 58, No. 4, 2005 Copyright © 2005 International Pediatric Research Foundation, Inc. Printed in U.S.A. Regulation of Myocardial Glucose Transporters GLUT1 and GLUT4 in Chronically Anemic Fetal Lambs J. CARTER RALPHE, PETER N. NAU, CHRISTOPHER E. MASCIO, JEFFREY L. SEGAR, AND THOMAS D. SCHOLZ Department of Pediatrics [J.C.R., P.N.N., J.L.S., T.D.S.], Department of Surgery [C.E.M.], University of Iowa, Iowa City, Iowa 52242 ABSTRACT Little is known about the chronic adaptations that take place steady state, GLUT4 protein localized to the sarcolemma mem- in the fetal heart to allow for increased substrate delivery in brane. These findings suggest that the glucose transporters are response to chronic stress. Because glucose is an important fuel post-transcriptionally regulated in myocardium of chronically for the fetal cardiomyocytes, we hypothesized that myocardial anemic fetal sheep with changes that mimic normal postnatal glucose transporters 1 and 4 (GLUT1 and GLUT4, respectively) development. Unlike the postnatal heart, localization of GLUT4 are up-regulated in the fetal sheep heart that is chronically to the cell membrane suggests the importance of GLUT4 in basal stressed by anemia. Fetal sheep at 128 d gestation underwent glucose uptake in the stressed fetal heart. (Pediatr Res 58: daily isovolumic hemorrhage and determination of myocardial 713–718, 2005) blood flow, oxygen consumption, and substrate utilization. At the endof3or7dofanemia, myocardial levels of GLUT1 and Abbreviations GLUT4 mRNA and protein were measured and subcellular ERK, extracellular-regulated kinase localization was determined. Despite stable heart rate and blood GLUT1(4), glucose transporter 1 (4) pressure, anemia caused a nearly 4-fold increase in right and left HIF-1␣, hypoxia-inducible factor 1␣ ventricular (RV and LV) free wall blood flow.
    [Show full text]
  • Regulation of Skeletal Muscle Glucose Transport and Glucose Metabolism by Exercise Training
    nutrients Review Regulation of Skeletal Muscle Glucose Transport and Glucose Metabolism by Exercise Training Parker L. Evans 1,2,3, Shawna L. McMillin 1,2,3 , Luke A. Weyrauch 1,2,3 and Carol A. Witczak 1,2,3,4,* 1 Department of Kinesiology, East Carolina University, Greenville, NC 27858, USA; [email protected] (P.L.E.); [email protected] (S.L.M.); [email protected] (L.A.W.) 2 Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, NC 27834, USA 3 East Carolina Diabetes & Obesity Institute, East Carolina University, Greenville, NC 27834, USA 4 Department of Biochemistry & Molecular Biology, Brody School of Medicine, East Carolina University, Greenville, NC 27834, USA * Correspondence: [email protected]; Tel.: +1-252-744-1224 Received: 8 September 2019; Accepted: 8 October 2019; Published: 12 October 2019 Abstract: Aerobic exercise training and resistance exercise training are both well-known for their ability to improve human health; especially in individuals with type 2 diabetes. However, there are critical differences between these two main forms of exercise training and the adaptations that they induce in the body that may account for their beneficial effects. This article reviews the literature and highlights key gaps in our current understanding of the effects of aerobic and resistance exercise training on the regulation of systemic glucose homeostasis, skeletal muscle glucose transport and skeletal muscle glucose metabolism. Keywords: aerobic exercise; blood glucose; functional overload; GLUT; hexokinase; insulin resistance; resistance exercise; SGLT; type 2 diabetes; weightlifting 1. Introduction Exercise training is defined as planned bouts of physical activity which repeatedly occur over a duration of time lasting from weeks to years.
    [Show full text]
  • Effect of Hydrolyzable Tannins on Glucose-Transporter Expression and Their Bioavailability in Pig Small-Intestinal 3D Cell Model
    molecules Article Effect of Hydrolyzable Tannins on Glucose-Transporter Expression and Their Bioavailability in Pig Small-Intestinal 3D Cell Model Maksimiljan Brus 1 , Robert Frangež 2, Mario Gorenjak 3 , Petra Kotnik 4,5, Željko Knez 4,5 and Dejan Škorjanc 1,* 1 Faculty of Agriculture and Life Sciences, University of Maribor, Pivola 10, 2311 Hoˇce,Slovenia; [email protected] 2 Veterinary Faculty, Institute of Preclinical Sciences, University of Ljubljana, Gerbiˇceva60, 1000 Ljubljana, Slovenia; [email protected] 3 Center for Human Molecular Genetics and Pharmacogenomics, Faculty of Medicine, University of Maribor, Taborska 8, 2000 Maribor, Slovenia; [email protected] 4 Department of Chemistry, Faculty of Medicine, University of Maribor, Taborska 8, 2000 Maribor, Slovenia; [email protected] (P.K.); [email protected] (Ž.K.) 5 Laboratory for Separation Processes and Product Design, Faculty of Chemistry and Chemical Engineering, University of Maribor, Smetanova 17, 2000 Maribor, Slovenia * Correspondence: [email protected]; Tel.: +386-2-320-90-25 Abstract: Intestinal transepithelial transport of glucose is mediated by glucose transporters, and affects postprandial blood-glucose levels. This study investigates the effect of wood extracts rich in hydrolyzable tannins (HTs) that originated from sweet chestnut (Castanea sativa Mill.) and oak (Quercus petraea) on the expression of glucose transporter genes and the uptake of glucose and HT constituents in a 3D porcine-small-intestine epithelial-cell model. The viability of epithelial cells CLAB and PSI exposed to different HTs was determined using alamarBlue®. qPCR was used to analyze the gene expression of SGLT1, GLUT2, GLUT4, and POLR2A. Glucose uptake was confirmed Citation: Brus, M.; Frangež, R.; by assay, and LC–MS/ MS was used for the analysis of HT bioavailability.
    [Show full text]
  • Transport of Sugars
    BI84CH32-Frommer ARI 29 April 2015 12:34 Transport of Sugars Li-Qing Chen,1,∗ Lily S. Cheung,1,∗ Liang Feng,3 Widmar Tanner,2 and Wolf B. Frommer1 1Department of Plant Biology, Carnegie Institution for Science, Stanford, California 94305; email: [email protected] 2Zellbiologie und Pflanzenbiochemie, Universitat¨ Regensburg, 93040 Regensburg, Germany 3Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305 Annu. Rev. Biochem. 2015. 84:865–94 Keywords First published online as a Review in Advance on glucose, sucrose, carrier, GLUT, SGLT, SWEET March 5, 2015 The Annual Review of Biochemistry is online at Abstract biochem.annualreviews.org Soluble sugars serve five main purposes in multicellular organisms: as sources This article’s doi: of carbon skeletons, osmolytes, signals, and transient energy storage and as 10.1146/annurev-biochem-060614-033904 transport molecules. Most sugars are derived from photosynthetic organ- Copyright c 2015 by Annual Reviews. isms, particularly plants. In multicellular organisms, some cells specialize All rights reserved in providing sugars to other cells (e.g., intestinal and liver cells in animals, ∗ These authors contributed equally to this review. photosynthetic cells in plants), whereas others depend completely on an ex- Annu. Rev. Biochem. 2015.84:865-894. Downloaded from www.annualreviews.org ternal supply (e.g., brain cells, roots and seeds). This cellular exchange of Access provided by b-on: Universidade de Lisboa (UL) on 09/05/16. For personal use only. sugars requires transport proteins to mediate uptake or release from cells or subcellular compartments. Thus, not surprisingly, sugar transport is criti- cal for plants, animals, and humans.
    [Show full text]
  • Distribution of Glucose Transporters in Renal Diseases Leszek Szablewski
    Szablewski Journal of Biomedical Science (2017) 24:64 DOI 10.1186/s12929-017-0371-7 REVIEW Open Access Distribution of glucose transporters in renal diseases Leszek Szablewski Abstract Kidneys play an important role in glucose homeostasis. Renal gluconeogenesis prevents hypoglycemia by releasing glucose into the blood stream. Glucose homeostasis is also due, in part, to reabsorption and excretion of hexose in the kidney. Lipid bilayer of plasma membrane is impermeable for glucose, which is hydrophilic and soluble in water. Therefore, transport of glucose across the plasma membrane depends on carrier proteins expressed in the plasma membrane. In humans, there are three families of glucose transporters: GLUT proteins, sodium-dependent glucose transporters (SGLTs) and SWEET. In kidney, only GLUTs and SGLTs protein are expressed. Mutations within genes that code these proteins lead to different renal disorders and diseases. However, diseases, not only renal, such as diabetes, may damage expression and function of renal glucose transporters. Keywords: Kidney, GLUT proteins, SGLT proteins, Diabetes, Familial renal glucosuria, Fanconi-Bickel syndrome, Renal cancers Background Because glucose is hydrophilic and soluble in water, lipid Maintenance of glucose homeostasis prevents pathological bilayer of plasma membrane is impermeable for it. There- consequences due to prolonged hyperglycemia or fore, transport of glucose into cells depends on carrier pro- hypoglycemia. Hyperglycemia leads to a high risk of vascu- teins that are present in the plasma membrane. In humans, lar complications, nephropathy, neuropathy and retinop- there are three families of glucose transporters: GLUT pro- athy. Hypoglycemia may damage the central nervous teins, encoded by SLC2 genes; sodium-dependent glucose system and lead to a higher risk of death.
    [Show full text]
  • Alterations in Net Glucose Uptake and in the Pancreatic B-Cell GLUT2 Transporter Induced by Diazoxide and by Secretory Stimuli
    291 Alterations in net glucose uptake and in the pancreatic B-cell GLUT2 transporter induced by diazoxide and by secretory stimuli L Zhao, Z Li, M Kullin,LAHBorg1 and F A Karlsson Department of Medical Sciences, University Hospital, SE-751 85 Uppsala, Sweden 1Department of Medical Cell Biology, University of Uppsala, PO Box 571, SE-751 23 Uppsala, Sweden (Requests for offprints should be addressed to L A H Borg; Email: [email protected]) Abstract The pancreatic B-cell GLUT2 transporter and glucose which may reflect a lowered energy requirement. Con- metabolism were examined in isolated rat islets subjected versely, islets subjected to a stimulated insulin secretion to treatments affecting insulin secretion. Diazoxide was with glipizide or a high extracellular K+ concentration used to inhibit, while glipizide or depolarization of the showed a reduced staining of the GLUT2 transporter. The plasma membrane with a high extracellular K+ concen- net glucose uptake and glucose oxidation were also tration were used to stimulate insulin release in short-term reduced. In islets exposed to the high K+ concentration no experiments. Islet GLUT2 and insulin were determined change in the molecular weight or phosphorylation of by quantitative immunohistochemistry and GLUT2 was GLUT2 was observed but a lesser amount of the trans- also determined by Western blot analysis. Islet net glucose porter was found by Western blot analysis. Thus, GLUT2 uptake and glucose oxidation were measured using radio- and glucose uptake in the pancreatic B-cell are modified actively labelled glucose. Exposure of the islets to diaz- by the secretory process, which suggests that changes in oxide was associated with a marked increase in the B-cell the glucose transporter have a functional role in normal plasma membrane staining for GLUT2 and increased net B-cell physiology.
    [Show full text]
  • IGF-I Increases the Recruitment of GLUT4 and GLUT3 Glucose
    European Journal of Endocrinology (2008) 158 361–366 ISSN 0804-4643 CLINICAL STUDY IGF-I increases the recruitment of GLUT4 and GLUT3 glucose transporters on cell surface in hyperthyroidism George Dimitriadis1, Eirini Maratou2, Eleni Boutati1, Anastasios Kollias1, Katerina Tsegka1, Maria Alevizaki3, Melpomeni Peppa1, Sotirios A Raptis1,2 and Dimitrios J Hadjidakis1 1Second Department of Internal Medicine, Research Institute and Diabetes Center,University General Hospital ‘Attikon’, Athens University, 1 Rimini Street, 12462 Haidari, Greece, 2Hellenic National Center for Research, Prevention and Treatment of Diabetes Mellitus and its Complications, 10675 Athens, Greece and 3Department of Clinical Therapeutics, 11528 Athens University, Athens, Greece (Correspondence should be addressed to G Dimitriadis; Email: [email protected], [email protected]) Abstract Objective: In hyperthyroidism, tissue glucose disposal is increased to adapt to high energy demand. Our aim was to examine the regulation of glucose transporter (GLUT) isoforms by IGF-I in monocytes from patients with hyperthyroidism. Design and methods: Blood (20 ml) was drawn from 21 healthy and 10 hyperthyroid subjects. The abundance of GLUT isoforms on the monocyte plasma membrane was determined in the absence and presence of IGF-I (0.07, 0.14, and 0.7 nM) using flow cytometry. Anti-CD14-phycoerythrin monocional antibody was used for monocyte gating. GLUT isoforms were determined after staining the cells with specific antisera to GLUT3 and GLUT4. Results: In monocytes from the euthyroid subjects, IGF-I increased the abundance of GLUT3 and GLUT4 on the monocyte surface by 25 and 21% respectively (P!0.0005 with repeated measures ANOVA). Hyperthyroidism increased the basal monocyte surface GLUT3 and GLUT4; in these cells, IGF-I had a marginal but highly significant effect (PZ0.003, with repeated measures ANOVA) on GLUT3 (11%) and GLUT4 (10%) translocation on the plasma membrane.
    [Show full text]
  • Sodium-Coupled Secondary Transporters 11 Insights from Structure-Based Computations
    b1151_Chapter-11.qxd 5/5/2011 12:27 PM Page 199 b1151 Molecular Machines FA Sodium-coupled Secondary Transporters 11 Insights from Structure-based Computations Elia Zomot, Ahmet Bakan, Indira H. Shrivastava, Jason DeChancie, Timothy R. Lezon and Ivet Bahar 1. Introduction: Biological Function and Classification The biological membrane bilayer is impermeable to almost all polar or charged molecules. In order for the various solutes to cross this barrier, integral membrane proteins have evolved to provide a hydrophilic environment within the membrane that can bind and translocate these solutes into or out of the cell, often against their electrochemical gradient. These transporters are conventionally classified into three classes on the basis of the energy source used for transport: (1) primary active transporters rely on light, hydrolysis of ATP or redox reactions, (2) secondary active transporters require the electrochemical gradient of ions across the membrane to power the “uphill” transloca- tion of the substrate, and (3) precursor/product antiporters exchange one molecule with its metabolic product independent of another source of energy.1,2 A major family of secondary transporters involves sodium- (and less often proton-) depend- ent symporters that couple the energy-costly translocation of the solute into the cell to that of sodium down its electrochemical gradient. These sodium-coupled transporters are found in all species and participate in a myriad biological functions, e.g. maintenance of efficient neuro- transmission,3,4,5 absorption of nutrients in the intestine,6 regulation of pH and cytoplasmic [Na+]7,8 and osmoregulation9,10 are only some of their physiological functions. Of particular interest among sodium-coupled symporters are two families: the dicarboxylate/ amino-acid:cation symporters (DAACS) and the neurotransmitter sodium symporters (NSS).
    [Show full text]
  • REVIEW the Molecular Basis of Insulin
    1 REVIEW The molecular basis of insulin-stimulated glucose uptake: signalling, trafficking and potential drug targets Sophie E Leney and Jeremy M Tavare´ Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK (Correspondence should be addressed to J M Tavare´; Email: [email protected]) Abstract The search for the underlying mechanism through which GLUT4 translocation and will attempt to address the spatial insulin regulates glucose uptake into peripheral tissues has relationship between the signalling and trafficking com- unveiled a highly intricate network of molecules that function ponents of this event. We will also explore the degree to in concert to elicit the redistribution or ‘translocation’ of which components of the insulin signalling and GLUT4 the glucose transporter isoform GLUT4 from intracellular trafficking machinery may serve as potential targets for membranes to the cell surface. Following recent technological the development of orally available insulin mimics for the advances within this field, this review aims to bring together treatment of diabetes mellitus. the key molecular players that are thought to be involved in Journal of Endocrinology (2009) 203, 1–18 Introduction Levine & Goldstein 1958). However, the mechanism by which insulin is able to stimulate glucose uptake was not Glucose homeostasis and diabetes mellitus elucidated until the early 1980s when two independent groups demonstrated that insulin promoted the movement of The ability of insulin to stimulate glucose uptake into muscle and adipose tissue is central to the maintenance of whole- a ‘glucose transport activity’ from an intracellular membrane body glucose homeostasis. Autoimmune destruction of the pool to the plasma membrane (Cushman & Wardzala 1980, pancreatic b-cells results in a lack of insulin production Suzuki & Kono 1980).
    [Show full text]
  • Is Retinal Metabolic Dysfunction at the Center of the Pathogenesis of Age-Related Macular Degeneration?
    International Journal of Molecular Sciences Review Is Retinal Metabolic Dysfunction at the Center of the Pathogenesis of Age-related Macular Degeneration? Thierry Léveillard 1,*, Nancy J. Philp 2 and Florian Sennlaub 3 1 . Department of Genetics, Sorbonne Université, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, F-75012 Paris, France 2 . Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA; [email protected] 3 . Department of Therapeutics, Sorbonne Université, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, F-75012 Paris, France; fl[email protected] * Correspondence: [email protected]; Tel.: +33-1-5346-2548 Received: 21 December 2018; Accepted: 5 February 2019; Published: 11 February 2019 Abstract: The retinal pigment epithelium (RPE) forms the outer blood–retina barrier and facilitates the transepithelial transport of glucose into the outer retina via GLUT1. Glucose is metabolized in photoreceptors via the tricarboxylic acid cycle (TCA) and oxidative phosphorylation (OXPHOS) but also by aerobic glycolysis to generate glycerol for the synthesis of phospholipids for the renewal of their outer segments. Aerobic glycolysis in the photoreceptors also leads to a high rate of production of lactate which is transported out of the subretinal space to the choroidal circulation by the RPE. Lactate taken up by the RPE is converted to pyruvate and metabolized via OXPHOS. Excess lactate in the RPE is transported across the basolateral membrane to the choroid. The uptake of glucose by cone photoreceptor cells is enhanced by rod-derived cone viability factor (RdCVF) secreted by rods and by insulin signaling. Together, the three cells act as symbiotes: the RPE supplies the glucose from the choroidal circulation to the photoreceptors, the rods help the cones, and both produce lactate to feed the RPE.
    [Show full text]