THE GLUCOSE TRANSPORTERS THAT FACILITATE THE PASSAGE OF GLUCOSE ACROSS THE BLOOD-BRAIN BARRIER
Open Access
- Author:
- Devraj, Kavi
- Graduate Program:
- Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 02, 2009
- Committee Members:
- Ian Alexander Simpson, Dissertation Advisor/Co-Advisor
Ian Alexander Simpson, Committee Chair/Co-Chair
Dr David Antonetti, Committee Member
James Robert Connor, Committee Member
Dr Steven Abcouwer, Committee Member
Dr Anthony Carruthers, Committee Member
Tao Lu Lowe, Committee Member - Keywords:
- 2-D Electrophoresis
Cultured Endothelial Cells
Microvessels
Polarization
Glucose Transporters
Blood-Brain Barrier
Luminal
Abluminal - Abstract:
- Glucose is the obligate energy fuel for the mammalian brain which consumes approximately 20% of plasma glucose that is almost entirely oxidized to CO2 and H2O. The first step in the metabolism of glucose within the brain is its transport across the blood-brain barrier (BBB), which is comprised of microvascular endothelial cells joined together by specialized tight junctions and is supported by astroglia and pericytes. These endothelial cells are polarized with the luminal (blood-side) and abluminal (brain-side) membranes being composed of variety of proteins specific to each membrane. Glucose must traverse both membranes of the BBB before entering the brain, and this transport is thought to be mediated by GLUT-1 glucose transporters at the BBB. We have investigated GLUT-1 modifications and the presence of alternate transporters to explain the disparity in GLUT-1 levels and glucose transport activity in the luminal and abluminal membranes of the BBB observed in earlier studies. Utilizing 2-D electrophoresis and iTRAQ™ (isobaric tagging for relative and absolute quantitation) approaches, we demonstrate that GLUT-1 is present in post-translationally modified forms at these two BBB membranes. We show that phosphorylation is a potential modification that occurs primarily at the C-terminus and/or cytoplasmic loop (6-7) regions of GLUT-1. We have also investigated glucose transporter expression in vitro using cultured microvascular endothelial cells in an attempt to characterize the in vivo glucose transport mechanism. Our results show that the abluminal forms of GLUT-1 are lost when microvascular endothelial cells are placed in culture, precluding their use for transport studies as they are not reflective of the in vivo situation. We suggest that this results from a loss of polarization of the BBB in vitro as demonstrated by dramatic under-expression of abluminal marker proteins compared to luminal marker proteins. The current studies demonstrate the presence of GLUT-3, a high affinity, high capacity glucose transporter at the BBB using iTRAQ™, RT-PCR, and Western blots. Overall, we suggest that the modifications in GLUT-1 at the BBB along with the GLUT-3 transporters potentially regulate glucose entry into brain across this vital barrier in normal and pathological states such as hypoxia and hypoglycemia.