Potential mechanism of insulin action on glucose transport in the isolated rat adipose cell. Apparent translocation of intracellular transport systems to the plasma membrane.

Detailed studies of [3mcytochalasin B-binding and the inhibition of glucose transport by cytochalasin B have recently permitted characterization of a specific class of D-glucose-inhibitable cytochalasin B-binding sites in purified isolated rat adipose cell plasma membranes, identification of these sites as functional glucose transport systems, and direct demonstration that insulin stimulates glucose transport in intact cells primarily by increasing the number of these systems. An abbreviated [3H]cytochalasin B-binding assay is used here to examine the microsomal membrane fraction for D-glucose-inhibitable binding sites which could potentially represent those transport systems appearing in the plasma membranes in response to insulin. In a series of three experiments, membranes in the microsomal fraction prepared from cells which have been preincubated for 30 min in the absence of insulin contain 38 pmol of D-glucose-inhibitable cytochalasin B-binding sites per mg of membrane protein, or approximately 5-fold that observed in the plasma membrane fraction prepared from the same cells. However, in the presence of 7.0 nM (1000 microunits/ml) insulin during the preincubation period, the number of these sites in the microsomal membrane fraction is decreased to 21 pmol/mg of membrane protein, while that in the plasma membrane fraction is increased to 24 pmol/mg of membrane protein. The numbers of D-glucose-inhibitable cytochalasin Bbinding sites in the intact cell have been estimated from the 5'-nucleotidase and NADH-cytochrome c reductase specific activities of each membrane fraction and the original homogenates. Of a total of approximately 3.16 X 10" binding sites/cell in the basal cells, the plasma and microsomal membranes account for 0.35 X 10" and 2.81 X 10' binding sites/cell, respectively. While insulin does not alter the estimated total number of binding sites, that in the plasma membranes rises to 2.08 X 10' sites/cell (a net increase of 1.73 X 10" sites/cell) while that in the microsomal membranes decreases to 1-09 X 10" sites/cell (a net decrease of 1.72 X 10' sites/cell). The stoichiometric nature of this relationship further