Effect of glucose-6-phosphate and pH on glucose transport in skeletal muscle plasma membrane giant vesicles.

The effect of glucose-6-phosphate (G-6-P) and pH on glucose transport was studied in skeletal muscle plasma membrane giant vesicles containing GLUT4 but not GLUT1. Vesicles (average diameter 7.6 microns) were obtained by collagenase treatment of muscle. The vesicles were incubated with 10 mmol l-1 G-6-P and, after 0.5 and 2 h of incubation, the intravesicular G-6-P concentration was 0.93 +/- 0.4 mmol l-1 and 1.18 +/- 0.5 mmol l-1 (mean +/- SE, n = 4), respectively. In order to increase the intravesicular G-6-P concentration, 0.001% saponin was added during incubation, which increased the 2-h intravesicular G-6-P concentration to 4.57 +/- 1.0 mmol l-1 (n = 4). Initially, vesicles were used for glucose transport studies after 30 min of incubation with 10 mmol l-1 of G-6-P. There was no effect of G-6-P on either the affinity constant (Km) or maximal velocity (Vmax) of the glucose transport. Subsequently, vesicles were incubated for 2 h with 10 mmol l-1 of G-6-P and 0.001% saponin. Still no effect of G-6-P on glucose transport could be detected. In contrast, the rate of D-glucose transport was affected, when extravesicular pH was varied from 6.0 to 7.8. The maximum glucose transport rate was found at pH 7.2 and was decreased at both higher and lower pH. It is concluded that G-6-P has no effect on GLUT4 intrinsic activity in rat skeletal muscle plasma membrane. In contrast, GLUT4 intrinsic activity is sensitive to changes in pH.