Glucose uptake in astrocytes, basal cardiomyocytes, endothelial cells, erythrocytes and smooth muscle is mediated by GLUT1. In most tissues, glucose utilization is limited by glucose uptake and increases in cellular metabolic demand rapidly increase cell surface GLUT1 intrinsic activity or GLUT1 content. A slower, adaptive response also occurs in which GLUT1 expression increases. Sugar transport in endothelial cells and erythrocytes is much faster than metabolism yet these cells also show adaptive and/or rapid transport responses. The reason may be that GLUT1-mediated transport in these cells limits glucose utilization in other tissues protected by blood-tissue barriers (e.g. brain, peripheral nerve, myocardium, retina) and that GLUT1 is uniquely amenable to acute catalytic regulation. Blood-tissue barriers comprise endothelial cells connected by tight junctions. Glucose metabolism in protected tissues requires glucose transfer across the barrier by GLUT1-mediated, trans-cellular transport. Impaired barrier transport compromises tissue function causing apoptosis, seizures, focal neurologic deficits and coma and may have genetic, endocrine and pharmacologic origins. Long-term glycopenia disrupts development. This proposal represents our continuing efforts to understand GLUT1 catalytic regulation, its role in organismal homeostasis and the insights this brings to other Major Facilitator Superfamily transport proteins. Our long-term goal is to translate these insights into practical intervention in clinical glycopenia. GLUT1-mediated glucose uptake involves rapid, ATP-insensitive, glucose translocation through a membrane-spanning """"""""channel"""""""" into a """"""""cage"""""""" formed by GLUT1 cytoplasmic loop 6 and C-terminal domains. Sugar release from the cage into cytoplasm is much slower and is further inhibited by ATP which restructures GLUT1 loop 6, exofacial loop 7 and the C-terminus. These changes involve specific loop 6 and C-terminal lysine residues and convert the cage to one which now prefers ?-D-glucose 20-fold over a-D-glucose. H+ and AMP antagonize these changes. This mechanism may represent a fundamental regulatory mechanism available to GLUT1 in all cells.
Specific Aim 1 tests the hypothesis that cytoplasmic loop 8 is the ATP binding domain by ESI MS-MS analysis of purified GLUT1 covalently modified with photoreactive nucleotide analogs and by mutagenesis of identified, labeled amino acids.
Specific Aim 2 tests the hypothesis that the C-terminus and cytoplasmic loop 6 play a primary role in GLUT1 regulation by swapping GLUT1 loop 6 and C-terminal domains with equivalent sequence from ATP-insensitive GLUT3 &4 and testing constructs for loss of ATP-responsiveness.
Specific Aim 3 tests the hypothesis that ATP converts GLUT1 to a ?-sugar-preferring carrier and asks whether GLUT1 C-terminus-L6 interactions and/or ATP binding mediate specificity changes.
Specific Aim 4 tests the hypothesis that rapid up-regulation of erythrocyte and blood brain barrier endothelial cell sugar transport represent a single fundamental GLUT1 regulatory mechanism by comparison of acute hypoglycemic stimulation of bEnd3 cell sugar uptake with ATP-depletion-stimulated red cell transport.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK036081-24
Application #
7837595
Study Section
Cellular Aspects of Diabetes and Obesity Study Section (CADO)
Program Officer
Sechi, Salvatore
Project Start
1985-09-25
Project End
2013-05-31
Budget Start
2010-06-01
Budget End
2011-05-31
Support Year
24
Fiscal Year
2010
Total Cost
$321,750
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Biochemistry
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655
Ojelabi, Ogooluwa A; Lloyd, Kenneth P; De Zutter, Julie K et al. (2018) Red wine and green tea flavonoids are cis-allosteric activators and competitive inhibitors of glucose transporter 1 (GLUT1)-mediated sugar uptake. J Biol Chem 293:19823-19834
Lloyd, Kenneth P; Ojelabi, Ogooluwa A; Simon, Andrew H et al. (2018) Kinetic Basis of Cis- and Trans-Allostery in GLUT1-Mediated Sugar Transport. J Membr Biol 251:131-152
Lloyd, Kenneth P; Ojelabi, Ogooluwa A; De Zutter, Julie K et al. (2017) Reconciling contradictory findings: Glucose transporter 1 (GLUT1) functions as an oligomer of allosteric, alternating access transporters. J Biol Chem 292:21035-21046
Ojelabi, Ogooluwa A; Lloyd, Kenneth P; Simon, Andrew H et al. (2016) WZB117 (2-Fluoro-6-(m-hydroxybenzoyloxy) Phenyl m-Hydroxybenzoate) Inhibits GLUT1-mediated Sugar Transport by Binding Reversibly at the Exofacial Sugar Binding Site. J Biol Chem 291:26762-26772
Sage, Jay M; Carruthers, Anthony (2014) Human erythrocytes transport dehydroascorbic acid and sugars using the same transporter complex. Am J Physiol Cell Physiol 306:C910-7
De Zutter, Julie K; Levine, Kara B; Deng, Di et al. (2013) Sequence determinants of GLUT1 oligomerization: analysis by homology-scanning mutagenesis. J Biol Chem 288:20734-44
Vollers, Sabrina S; Carruthers, Anthony (2012) Sequence determinants of GLUT1-mediated accelerated-exchange transport: analysis by homology-scanning mutagenesis. J Biol Chem 287:42533-44
Cura, Anthony J; Carruthers, Anthony (2012) Role of monosaccharide transport proteins in carbohydrate assimilation, distribution, metabolism, and homeostasis. Compr Physiol 2:863-914
Cura, Anthony J; Carruthers, Anthony (2012) AMP kinase regulation of sugar transport in brain capillary endothelial cells during acute metabolic stress. Am J Physiol Cell Physiol 303:C806-14
Mangia, Silvia; DiNuzzo, Mauro; Giove, Federico et al. (2011) Response to 'comment on recent modeling studies of astrocyte-neuron metabolic interactions': much ado about nothing. J Cereb Blood Flow Metab 31:1346-53

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