COMPARTMENT ABLATION ANALYSIS OF THE INSULIN-RESPONSIVE GLUCOSE-TRANSPORTER (GLUT4) IN 3T3-L1 ADIPOCYTES

The translocation of a unique facilitative glucose transporter isoform (GLUT4) from an intracellular site to the plasma membrane accounts fo r the large insulin-dependent increase in glucose transport observed i n muscle and adipose tissue. The intracellular location of GLUT4 in th e basal state and the pathway by which it reaches the cell surface upo n insulin stimulation are unclear. Here, we have examined the colocali zation of GLUT4 with the transferrin receptor, a protein which is know n to recycle through the endosomal system. Using an anti-GLUT4 monoclo nal antibody we immunoisolated a vesicular fraction from an intracellu lar membrane fraction of 3T3-L1 adipocytes that contained > 90%, of th e immunoreactive GLUT4 found in this fraction, but only 40% of the tra nsferrin receptor (TfR). These results suggest only a limited degree o f colocalization of these proteins. Using a technique to cross-link an d render insoluble ('ablate') intracellular compartments containing th e TfR by means of a transferrin-horseradish peroxidase conjugate (Tf-H RP), we further examined the relationship between the endosomal recycl ing pathway and the intracellular compartment containing GLUT4 in thes e cells. Incubation of non-stimulated cells with Tf-HRP for 3 h at 37 degrees C resulted in quantitative ablation of the intracellular TfR, GLUT1 and mannose-6-phosphate receptor and a shift in the density of R ab5-positive membranes. In contrast, only 40% of intracellular GLUT4 w as ablated under the same conditions. Ablation was specific for the en dosomal system as there was no significant ablation of either TGN38 or 1gp120, which are markers for the trans Golgi reticulum and lysosomes respectively. Subcellular fractionation analysis revealed that most o f the ablated pools of GLUT4 and TfR were found in the intracellular m embrane fraction. The extent of ablation of GLUT4 from the intracellul ar fraction was unchanged in cells which were insulin-stimulated prior to ablation, whereas GLUT1 exhibited increased ablation in insulin-st imulated cells. Pretreatment of adipocytes with okadaic acid, an inhib itor of Type-I and -IIa phosphatases, increased GLUT4 ablation in the presence of insulin, consistent with okadaic acid increasing the inter nalization of GLUT4 from the plasma membrane under these conditions. U sing a combination of subcellular fractionation, vesicle immunoadsorpt ion and compartment ablation using the Tf-HRP conjugate we have been a ble to resolve overlapping but distinct intracellular distributions of the TfR and GLUT4 in adipocytes. At least three separate compartments were identified: TfR-positive/GLUT4-negative, TfR-negative/GLUT4-posi tive, and TfR-po- sitive/GLUT4-positive, as defined by the relative ab undance of these two markers. We propose that the TfR-negative/GLUT4-p ositive compartment, which contains approximately 60% of the intracell ular GLUT4, represents a specialized intracellular compartment that is withdrawn from the endosomal system. The biosynthesis and characteris tics of this compartment may be fundamental to the unique insulin regu lation of GLUT4.