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.