Abstract
The clinical use of several first generation HIV protease inhibitors (PIs) is associated with the development of insulin resistance. Indinavir has been shown to act as a potent reversible noncompetitive inhibitor of zero-trans glucose influx via direct interaction with the insulin responsive facilitative glucose transporter GLUT4. Newer drugs within this class have differing effects on insulin sensitivity in treated patients. GLUTs are known to contain two distinct glucose-binding sites that are located on opposite sides of the lipid bilayer. To determine whether interference with the cytoplasmic glucose binding site is responsible for differential effects of PIs on glucose transport, intact intracellular membrane vesicles containing GLUT1 and GLUT4, which have an inverted transporter orientation relative to the plasma membrane, were isolated from 3T3-L1 adipocytes. The binding of biotinylated ATB-BMPA, a membrane impermeable bis-mannose containing photolabel, was determined in the presence of indinavir, ritonavir, atazanavir, tipranavir, and cytochalasin b. Zero-trans 2-deoxyglucose transport was measured in both 3T3-L1 fibroblasts and primary rat adipocytes acutely exposed to these compounds. PI inhibition of glucose transport correlated strongly with the PI inhibition of ATB-BMPA/transporter binding. At therapeutically relevant concentrations, ritonavir was not selective for GLUT4 over GLUT1. Indinavir was found to act as a competitive inhibitor of the cytoplasmic glucose binding site of GLUT4 with a KI of 8.2 µM. These data establish biotinylated ATB-BMPA as an effective probe to quantify accessibility of the endofacial glucose-binding site in GLUTs and reveal that the ability of PIs to block this site differs among drugs within this class. This provides mechanistic insight into the basis for the clinical variation in drug-related metabolic toxicity.
Highlights
The development and clinical use of HIV protease inhibitors has greatly contributed to the transition of HIV infection from a once fatal disease to its current status as a chronic condition [1]
Greater than 70% of the GLUT4 vesicles could by immunoisolated from non-detergent solubilized low-density microsome (LDM) using a C-terminal directed GLUT4 antibody demonstrating that the vast majority of GLUT4 in LDM was found in this membrane orientation (Fig. 1A)
Initial experiments revealed that glucose transporters found in isolated LDM are photolabeled with biotinylated ATBBMPA in a LDM concentration dependent and cytochalasin B (CB) sensitive manner (Fig. 1B)
Summary
The development and clinical use of HIV protease inhibitors has greatly contributed to the transition of HIV infection from a once fatal disease to its current status as a chronic condition [1]. Tempering enthusiasm for this major advance in HIV treatment is the growing realization that patients treated with combined antiretroviral treatment regimens are at increased risk for the development of pro-atherogenic metabolic side effects including dyslipidemia and insulin resistance [2,3]. A direct correlation between the ability of these drugs to block glucose transport in vivo and effects on insulin sensitivity in treated patients has been established [8]. Insight into the molecular basis for PI-mediated insulin resistance may provide a basis for novel approaches to treating the growing worldwide epidemic of type 2 diabetes mellitus
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