Efforts to understand the mechanisms for altered glucose homeostasis in HIV infected patients have been limited by the complexity of interacting environmental, genetic, treatment and disease-related factors involved. Nevertheless, it is well established that antiretroviral therapy directly contributes to the development of diabetes 
. Among the various antiretroviral agents in clinical use, HIV protease inhibitors are known to influence peripheral glucose disposal, hepatic glucose production, and insulin secretion 
. Contrary to the initial speculation that induction of insulin resistance is a shared feature of all PIs, subsequent investigation has shown that individual agents within this drug class have differing effects on glucose homeostasis, both in vitro
and in treated patients. The initial identification and characterization of GLUT4 as a direct molecular target of PIs was performed using indinavir 
. The isoform selectivity of this drug (i.e. the ability to block GLUT4 activity with no effect on GLUT1) was established in Xenopus
oocytes heterologously expressing either of these glucose transporters 
. While it has been generally assumed that all PIs possess the same degree of isoform selectivity as indinavir, direct comparisons of glucose transport blockade in GLUT1 versus GLUT4 expressing cells have been lacking. The binding affinity (KI
) of indinavir for GLUT4 in the oocyte system (50 µM) differs from that observed in primary adipocytes (15 µM). While the basis for this difference is unknown, contributing factors may include subtle structural differences in the expressed transporter due to lipid composition, assay temperature, the presence of additional proteins, or other factors. It was therefore necessary to directly compare the ability of both first generation and newer PIs to alter GLUT1 versus GLUT4 activity. These data provide a more comprehensive assessment of similarities and differences in the behavior of these PIs on facilitative glucose transport.
Several observations related to the ability of PIs examined in this study to compete for endofacial ATB BMPA binding have direct relevance to understanding the metabolic toxicities of these drugs in antiretroviral treatment regimens. Importantly, few studies to date have directly assessed the relationship between intracellular PI concentrations and impaired glucose uptake. Whether PI import occurs via simple diffusion or through mediated transport, sufficient drug levels may be present within the cytosol even when serum levels are low 
. In addition, while it has been assumed that all PIs possess the same degree of GLUT isoform selectivity as indinavir, several PIs including ritonavir do not appear to distinguish among these transporters. Thus, the effects of some PIs on glucose homeostasis in tissues that do not express GLUT4 (such as hepatic glucose production in the liver and glucose-stimulated insulin secretion from β-cells) may still be mediated by changes in glucose transport. Comparison of the effects of various PIs in these tissues may provide further insight into the mechanistic basis for altered glucose homeostasis. More comprehensive assessment of the ability of individual PIs to block each of the other known GLUTs may provide insight into glucotoxicities. While atazanavir has a more favorable metabolic profile relative to first generation PIs, the current studies demonstrate that at drug levels above those typically achieved during clinical use, the potential for significantly altering glucose transport exists. The inability of tipranavir to alter either ATB BMPA binding or 2DG transport further supports the role of peptidomimetic structure in mediating binding to GLUTs.
Understanding of the molecular basis for the development of insulin resistance in HIV infected patients treated with PIs has already contributed to success in developing drugs within this class that do not directly alter glucose homeostasis. Nevertheless, many of these newer agents including tipranavir are associated with dyslipidemia 
and may therefore indirectly contribute to impaired insulin signaling. Furthermore, with the potential for development of viral resistance over time, the need for continued drug development remains. Characterization of the molecular interactions between candidate drugs and GLUTs will assist ongoing efforts for rationale drug design, not only for antiviral efficacy, but also for metabolic toxicity.
Beyond further understanding of the mechanisms for PI-mediated insulin resistance, the ability to distinguish compounds that selectively interact with GLUT4 from those that bind to both GLUT1 and GLUT4 suggests that it may be possible to identify small molecule inhibitors of each of the other known GLUTs 
. The availability of specific pharmacologic inhibitors of these transporters would provide a means to further characterize the functional role of these isoforms prior to the induction of potential compensatory changes in gene knockout models.
The development and use of an ATB-BMPA based assay for labeling of the cytoplasmic glucose binding site of GLUTs provides potential uses for this agent that extends its traditional use to quantify cell surface levels of GLUTs. This includes elucidation of the functional significance of cytosolic binding of known GLUT-interacting proteins and the discovery of additional protein-protein interactions. Such applications may aid efforts to identify means to improve diabetes treatment in the wider context of non-HIV associated insulin resistance.