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 ?-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. PROJECT NARRATIVE Gycopenia (tissue glucose shortage) can have genetic, endocrine and pharmacologic origins, results in seizures, focal neurologic deficits, coma and, if uncorrected, impairs development. This proposal continues our efforts to understand how the activity of the blood brain barrier glucose transport protein is regulated, its stabilizing role in organismal health and how these insights impact our understanding of the wider family of Major Facilitator Superfamily transport proteins. Our long-term goal is to translate these insights into practical intervention in clinical glycopenia.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK036081-26
Application #
8267043
Study Section
Cellular Aspects of Diabetes and Obesity Study Section (CADO)
Program Officer
Sechi, Salvatore
Project Start
1985-09-25
Project End
2013-11-30
Budget Start
2012-06-01
Budget End
2013-11-30
Support Year
26
Fiscal Year
2012
Total Cost
$318,533
Indirect Cost
$122,513
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
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Blodgett, David M; De Zutter, Julie K; Levine, Kara B et al. (2007) Structural basis of GLUT1 inhibition by cytoplasmic ATP. J Gen Physiol 130:157-68
Leitch, Jeffry M; Carruthers, Anthony (2007) ATP-dependent sugar transport complexity in human erythrocytes. Am J Physiol Cell Physiol 292:C974-86
Graybill, C; van Hoek, A N; Desai, D et al. (2006) Ultrastructure of human erythrocyte GLUT1. Biochemistry 45:8096-107
Cloherty, Erin K; Levine, Kara B; Graybill, Christopher et al. (2002) Cooperative nucleotide binding to the human erythrocyte sugar transporter. Biochemistry 41:12639-51
Levine, Kara B; Cloherty, Erin K; Hamill, Stephanie et al. (2002) Molecular determinants of sugar transport regulation by ATP. Biochemistry 41:12629-38
Cloherty, E K; Levine, K B; Carruthers, A (2001) The red blood cell glucose transporter presents multiple, nucleotide-sensitive sugar exit sites. Biochemistry 40:15549-61
Cloherty, E K; Hamill, S; Levine, K et al. (2001) Sugar transporter regulation by ATP and quaternary structure. Blood Cells Mol Dis 27:102-7

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