GSBS Dissertations and Theses

Approval Date

10-15-2010

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences, Biochemistry and Molecular Pharmacology

Subjects

Glucose Transporter Type 1; Endothelial Cells; Metabolism; Stress, Physiological; Dissertations, UMMS

Abstract

Studies have demonstrated that under conditions of chronic metabolic stress, GLUT1-mediated sugar transport is upregulated at the blood-brain barrier by a number of mechanisms. Although acute metabolic stress has also been shown to increase GLUT1-mediated transport, the mechanisms underlying this regulation remain unclear. This work attempts to explain how GLUT1-mediated sugar uptake is increased during acute metabolic stress, as well as explore the factors involved in this modulation of sugar transport in blood-brain barrier endothelial cells. Glucose depletion, KCN and FCCP were applied to brain microvascular endothelial cell line bEnd.3 in order to induce acute metabolic stress by ATP depletion. Kinetic sugar uptake measurements in combination with qPCR, whole cell lysate western blots, and cell-surface biotinylation were employed to probe for changes in GLUT1-mediated sugar uptake, GLUT1 expression levels, and GLUT1 localization during metabolic stress. Finally, the role of AMP-activated kinase (AMPK) in the bEnd.3 cell response to acute stress was examined using the specific AMPK activator AICAR and inhibitor Compound C.

The data presented in this thesis supports the following two conclusions: 1. GLUT1-mediated sugar transport in bEnd.3 cells during acute metabolic stress is increased 3-7 fold due to translocation of intracellular GLUT1 to the plasma membrane, with no change in expression of total GLUT1 protein, and 2. AMPK plays a direct role in modulating increases in GLUT1-mediated sugar transport in bEnd.3 cells during acute metabolic stress by regulating trafficking of GLUT1 to the plasma membrane.

Rights and Permissions

Copyright is held by the author, with all rights reserved.

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