Regulation of the Functional Expression of Hexose Transporter GLUT-1 by Glucose in Murine Fibroblasts: Role of Lysosomal Degradation

The nature of the membrane compartments involved in the regulation by glucose of hexose transport is not well defined. The effect of inhibitors of lysosomal protein degradation on hexose transport (i.e., uptake of [³H]-2-deoxy-D-glucose) and hexose transporter protein GLUT-1 (i.e., immunoblotting with antipeptide serum) in glucose-fed and -deprived cultured murine fibroblasts (3T3-C2 cells) was studied. The acidotropic amines chloroquine (20 µM) and ammonium chloride (10 mM) cause accumulation (both ~ 4-fold) of GLUT-1 protein and a small increase (both ~25%) in hexose transport in glucose-fed fibroblasts (24 h). The endopeptidase inhibitor, leupeptin (100 µM) causes accumulation (~ 4-fold) of GLUT-1 protein in glucose-fed fibroblasts (24 h) without changing hexose transport (≤5%). These agents do not greatly alter the electrophoretic mobility of GLUT-1. Neither chloroquine nor leupeptin augment the glucose deprivation (24 h) induced increases in hexose transport (~4-fold) and GLUT-1 content (~7-fold). In contrast, chloroquine or leupeptin diminish the reversal by glucose refeeding of the glucose deprivation induced accumulation of GLUT-1 protein but fail to alter the return of hexose transport to control levels. These results with inhibitors of lysosomal function are consistent with a model in which for murine fibroblasts (i) degradation of GLUT-1 occurs by routing the carrier through acidified compartments to the lysosomes for proteolysis; (ii) inhibition of lysosomal degradation and buffering of acidified intracellular membrane compartments do not cause changes in the glycosylation of GLUT-1; (iii) degradation of GLUT-1 may be indirectly inhibited in glucose-deprived cells; (iv) degradation of GLUT-1 does not directly control its functional expression; and (v) the ability of glucose to downregulate the functional expression of GLUT-1 may involve“internalization”into compartments which are independent of the pathways involved in its lysosomal degradation.