A structural basis for the acute effects of HIV protease inhibitors on GLUT4 intrinsic activity

Abstract

Human immunodeficiency virus (HIV) protease inhibitors (PIs) act as reversible noncompetitive inhibitors of GLUT4 with binding affinities in the low micromolar range and are known to contribute to alterations in glucose homeostasis during treatment of HIV infection. As aspartyl protease inhibitors, these compounds all possess a core peptidomimetic structure together with flanking hydrophobic moieties. To determine the molecular basis for GLUT4 inhibition, a family of related oligopeptides containing structural elements found in PIs was screened for their ability to inhibit 2-deoxyglucose transport in primary rat adipocytes. The peptide oxybenzylcarbonyl-His-Phe-Phe-O-ethyl ester (zHFFe) was identified as a potent inhibitor of zero-trans glucose flux with a K(i) of 26 mum. Similar to PIs, transport inhibition by this peptide was acute, noncompetitive, and reversible. Within a Xenopus oocyte expression system, zHFFe acutely and reversibly inhibited GLUT4-mediated glucose uptake, whereas GLUT1 activity was unaffected at concentrations as high as 1 mm. The related photoactivatable peptide zHFF-p-benzoylphenylalanine-[(125)I]Tyr-O-ethyl ester selectively labeled GLUT4 in rat adipocytes and indinavir effectively protected against photolabeling. Furthermore, GLUT4 bound to a peptide affinity column containing the zHFF sequence and was eluted by indinavir. These data establish a structural basis for PI effects on GLUT4 activity and support the direct binding of PIs to the transport protein as the mechanism for acute inhibition of insulin-stimulated glucose uptake.

FIG. 1

Structure features of HIV protease inhibitors. The core peptidomimetic structure found within all HIV protease inhibitors is shown in the shaded regions. A, amprenavir; B, indinavir; C, ritonavir; D, lopinavir; E, nelfinavir; F, saquinavir; G, atazanavir.

FIG. 2

Inhibition of 2-deoxyglucose uptake by aromatic peptides in primary rat adipocytes. Aromatic peptides (200 μM) were added 1 min prior to measuring [³H]2-deoxyglucose transport activity (50 μM, 1 min at 37 °C). All assays were performed in triplicate and are expressed as the percent of a vehicle-treated control ±S.E. Asterisks (*) indicate p < 0.05 as determined by an analysis of variance.

FIG. 3

Kinetic analysis of GLUT4 inhibition by z-His-Phe-Phe-O-Et. A, the uptake of [³H]2-deoxyglucose (◆, 50μM; ■, 100μM; ▲, 200 μM) was measured in Xenopus oocytes 3 days after injecting GLUT4 mRNA. The peptide zHFFe was added 6 min prior to the start of each 30-min assay performed at 22 °C. B, [³H]2-deoxyglucose uptake was measured in insulin-stimulated primary rat adipocytes 1 min following the addition of zHFFe. Assays were carried out for 1 min at 37 °C. The data are expressed as Dixon plots where the vertical axis represents the reciprocal of the transport velocity. All points represent the mean ± S.E. of three determinations.

FIG. 4

Reversible isoform-selective inhibition of GLUT4 activity by z-His-Phe-Phe-O-Et.A, [³H]2-deoxyglucose uptake (30 min at 22 °C) was measured 3 days following the injection of mRNA for each GLUT isoform into stage 5 Xenopus oocytes. The zHFFe peptide (dis-solved in dimethylformamide) was added 6 min prior to initiating the uptake assay. An equivalent volume of vehicle was added to the zero peptide control. Results represent the mean ± S.E. of three independent experiments. Asterisk (*) indicates p < 0.05. B, oocytes (15-20/group) were incubated for 60 s with 200 μM of z-His-Phe-Phe-O-Et (Peptide + Wash) or Bath's saline (Control). Oocytes were then either washed with peptide-free Barth's saline (Wash) or incubated in the continued presence of peptide (Peptide) during the 2-deoxyglucose up-take assay. Results represent the mean ± S.E.; asterisk (*) indicates p < 0.01 versus control.

FIG. 5

Inhibition of glucose uptake in rat adipocytes with z-His-Phe-Phe-Bpa-[125I]Tyr-O-Et. A, the indicated concentrations of peptide were added to insulin-stimulated primary rat adipocytes 1 min prior to measuring [³H]2-deoxyglucose uptake (1 min at 37 °C). HFF and CB represent positive controls in which 200 μM of the peptide zHFFe or 20 μM cytochalasin b, respectively, were added to the assay mixture. B, insulin-stimulated primary rat adipocytes were treated with the indicated concentrations of peptide and exposed to 254 nm light (black bars) or kept in the dark (gray bars) for 3 min at 37 °C. The adipocytes were then immediately washed three times with KRB to remove unreacted peptide before measuring residual 2-deoxyglucose transport activity. All assays were performed in triplicate and the values shown represent the mean ± S.E. Asterisks (*) indicate p < 0.05 compared with untreated control as determined by the Student's t test.

FIG. 6

Photoaffinity labeling of GLUT4 with z-His-Phe-Phe-Bpa-[¹²⁵I]Tyr-O-Et. A, SDS-PAGE analysis of primary rat adipocytes treated with 4 μM of photoactivated z-His-Phe-Phe-Bpa-[¹²⁵I]Tyr-O-Et in the presence or absence of 500 μM indinavir. Total cell homogenates (10 μg/lane) were analyzed by Western blot analysis with polyclonal antibody directed toward the carboxyl terminus of GLUT4 (Western) or autoradiography (Autorad) to determine incorporated ¹²⁵I label. B, the total cellular homogenates from photolabeled rat adipocytes were immunoprecipitated using affinity-purified polyclonal antibody directed toward partially purified GLUT4 and subjected to either Western blot analysis using the GLUT4 carboxyl-terminal antibody or autoradiography. Each lane contains 20 μg of protein.

FIG. 7

Peptide affinity chromatography of GLUT4. Primary rat adipocytes homogenized and passed over a 1-ml peptide affinity column containing the peptide z-His-Phe-Phe-Phe-Lys linked via the lysine residue to an agarose resin. Shown is a Western blot of column fractions analyzed using polyclonal antibody directed toward the carboxyl terminus of rat GLUT4. Lane 1 represents the adipocyte homogenate (5 μg) prior to loading onto the peptide column. Column wash fractions are shown in lanes 2-4, and protein eluted with buffer containing 500 μM indinavir is shown in lanes 5-8 (50 μl/lane). Rat GLUT4 heterologously expressed in Xenopus oocytes is shown in lane C as a positive control.