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Projects
The long-term objective of this project is to
understand the molecular mechanisms of facilitative glucose
transport. The goal of our current studies is to elucidate the
structural biochemistry determining transmembrane transport of
glucose by the human erythrocyte transporter GLUT1 and the
insulin-responsive glucose transporter GLUT4. The positioning of
the GLUT transmembrane alpha-helices which form the boundaries of
the aqueous glucose permeation pathway are being examined by
chemically cross-linking engineered paired sulfhydryl residues
within a cysteine-less GLUT1 transporter heterologously expressed
in Xenopus ooctyes. To facilitate structural analyses, mutant
GLUT1 transporters are being over-expressed in insect cells,
purified and functionally reconstituted into lipid vesicles.
Inter-helical distances and dynamic movement upon the addition of
specific GLUT inhibitors are being determined by site-directed
spin-labeling and electron paramagnetic resonance spectroscopy.
Taken together, these studies are providing a detailed structural
basis for our understanding of glucose transport. We anticipate
that this will allow the development of novel glucose
transporter-directed therapies to control glucose homeostasis in
human disease.
The use of HIV protease inhibitors (PIs) has
been associated with several metabolic changes including
lipodystrophy, hyperlipidemia and insulin resistance. Our
laboratory has recently discovered that PIs are capable of
selectively inhibiting GLUT4, the major insulin responsive glucose
transporter. Our current objective is to determine the mechanism
by which this inhibition occurs. We are using several approaches
to achieve this goal. First, careful kinetic analysis of the
inhibition process is being conducted in primary rat adipocytes
and in Xenopus oocytes heterologously expressing GLUT4. The site
of HIV protease inhibitor binding to GLUT4 is also being
determined through photolabeling of the transporter in Xenopus
oocytes and/or 3T3-L1 adipocytes using synthesized reactive
retroviral protease inhibitor derivatives. Finally, the ability
of HIV protease inhibitors to acutely and reversibly cause insulin
resistance in vivo is being investigated by measuring glucose
disposal under euglycemic hyperinsulinemic clamp conditions in
both normal and diabetes susceptible rodents. A better
understanding of the mechanism by which the activity of
facilitative glucose transporters can be acutely modulated in an
isoform specific manner will provide a new means of studying
glucose homeostasis in normal individuals and those with disorders
of glucose regulation such as diabetes mellitus. The results of
this research will also facilitate the development of newer HIV
protease inhibitors that maintain their efficacy in HIV treatment
while avoiding their adverse metabolic consequences.
The long-term objective of this project is to
understand the role of facilitative glucose transport in
myocardial energy homeostasis. While the healthy heart primarily
relies upon fatty acid catabolism for basal energy needs, glucose
provides a significant source of fuel during periods of acute
stress. Compensatory changes in glucose transporter expression in
response to the chronic induction of insulin resistance have
limited the ability to draw definitive conclusions from genetic
and environmental models of diabetes. Using acute, selective, and
reversible inhibitors of GLUTs, we are investigating the
functional role of facilitative glucose transport in normal and
diseased rodent myocardium. Studies are being conducted in vivo
(using mouse models of cardiac injury and failure), in situ (using
the isolated working heart model), and in vitro (using cultured
cardiomyocytes). These studies are providing significant insights
into the functional role of GLUT4 in meeting the energy demands of
the heart under basal and stressed conditions. This work is also
providing evidence for direct effects of HIV protease inhbitors on
cardiovascular morbidity in patients receiving these drugs.
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SCHOOL OF MEDICINE
