Mechanism of insulin action on glucose transport in the isolated rat adipose cell. Enhancement of the number of functional transport systems

[³H]Cytochalasin B binding and its competitive inhibition by D-glucose transport systems in plasma membranes prepared from isolated rat epididymal adipose cells. Curve-fitting analysis of equilibrium cytochalasin B binding in the absence and presence of 500 mM D-glucose permits identification of 3 classes of binding sites, only one of which is inhibited by D-glucose. Cytochalasin E reduces cytochalasin B binding to those sites which are not sensitive to D-glucose inhibition, thereby permitting a more precise determination of the characteristics of the D-glucose-sensitive site. This site comprises less than 25% of the total number of sites and binds cytochalasin B with a dissociation constant of approximately 120 nM. Since the inhibition constant of cytochalasin B for D-glucose uptake by adipose cell plasma membranes is similar in value, this site is tentatively identified as the glucose transport system. Plasma membranes prepared from isolated adipose cells which have not been preincubated with insulin bind approximately 4 pmol of cytochalasin B/mgof membrane protein to the D-glucose-inhibitable binding site. If 7.0 nM (1000 microunits/ml) insulin is present during preincubation, cytochalasin B binding to the plasma membranes is increased 4-fold without alterations either in this site's dissociation constant or in the characteristics of the other two cytochalasin B binding sites. These results suggest that insulin stimulates glucose transport in isolated rat adipose cells by activating identical, but basally inactive, glucose transport systems.