Submitted on April 7, 2006
Accepted on February 19, 2007
Infusion of a Biotinylated Bis-Glucose Photolabel: A New Method to Quantify Cell-Surface GLUT4 in the Intact Mouse Heart
Edward J. Miller1, Ji Li2, Kevin M. Sinusas2, Geoffrey D. Holman3, and Lawrence H. Young2*
1 Cardiovascular Medicine, Yale University Schoolf of Medicine, New Haven, Connecticut, United States
2 Cardiovascular Medicine, Yale University School of Medicine, New Haven, Connecticut, United States
3 Department of Biology and Biochemistry, University of Bath, Bath, Avon, United Kingdom
* To whom correspondence should be addressed. E-mail: lawrence.young{at}yale.edu.
Glucose uptake in the heart is mediated by specific glucose transporters (GLUTs) present on cardiomyocyte cell surface membranes. Metabolic stress and insulin both increase glucose transport by stimulating the translocation of glucose transporters from intracellular storage vesicles to the cell surface. Isolated perfused transgenic mouse hearts are commonly used to investigate the molecular regulation of heart metabolism, however, current methods to quantify cell-surface glucose transporter content in intact mouse hearts are limited. Therefore, we developed a novel technique to directly assess the cell-surface content of the cardiomyocyte glucose transporter GLUT4 in perfused mouse hearts, using a cell-surface impermeant biotinylated bis-glucose photolabeling reagent (bio-LC-ATB-BGPA). Bio-LC-ATB-BGPA was infused through the aorta and cross-linked to cell-surface GLUTs. Bio-LC-ATB-BGPA-labeled GLUT4 was recovered from cardiac membranes by streptavidin-isolation and quantified by immunoblotting. Bio-LC-ATB-BGPA-labeling of GLUT 4 was saturable and competitively inhibited by D-glucose. Stimulation of glucose uptake by insulin in the perfused heart was associated with parallel increases in bio-LC-ATB-BGPA-labeling of cell-surface GLUT4. Bio-LC-ATB-BGPA also labeled cell-surface GLUT1 in the perfused heart. Thus, photolabeling provides a novel approach to assess cell-surface glucose transporter content in the isolated perfused mouse heart and may prove useful to investigate the mechanisms through which insulin, ischemia and other stimuli regulate glucose metabolism in the heart and other perfused organs.
