Genistein inhibits insulin-stimulated glucose transport and decreases immunocytochemical labeling of GLUT4 carboxyl-terminus without affecting translocation of GLUT4 in isolated rat adipocytes: additional evidence of GLUT4 activation by insulin.

A recent study from this laboratory (Abler et al., J. Biol. Chem. 267, 18172-18179, 1992) showed genistein blocked insulin-stimulated glucose oxidation without affecting receptor autophosphorylation or tyrosine kinase activity. The mechanism by which genistein inhibited insulin-stimulated glucose metabolism was investigated in the present study. Insulin caused a approximately 12-fold increase in 3-O-methyl-D-glucose (3OMG) uptake compared to that of control cells. Basal and insulin-stimulated 3OMG transport was inhibited 40-60% by genistein in a concentration-dependent manner (10-100 micrograms/ml). Genistein had no effect on insulin-stimulated GLUT4 translocation from low density microsomes to plasma membranes as determined by Western blotting. These results suggested that genistein inhibited glucose transport in adipocytes by decreasing the intrinsic activity, rather than the number, of the plasma membrane-associated glucose transporters. We also previously reported that insulin treatment of adipocytes resulted in the immunocytochemically visualized unmasking of the carboxyl-terminus of plasma membrane-associated GLUT4 and suggested the unmasking might be related to an insulin-induced increase in the intrinsic activity of the glucose transporter (Smith et al., Proc. Natl. Acad. Sci. USA 88, 6893-6897, 1991). In the present study, genistein decreased immunocytochemical labeling of plasma membrane-associated GLUT4 by approximately 50% in control and insulin-treated adipocytes by carboxyl-terminus antibodies but had no effect on labeling observed in an amino-terminus antibody. Since genistein did not affect the number of plasma membrane-associated GLUT4 transporters, this result supports the hypothesis that conformational changes in the glucose transporter, reflected by the ability of anti-carboxyl-terminus antibodies to bind to the transporter, may be an indication of the intrinsic activity of the plasma membrane-associated transporter. We therefore conclude that conformational changes in and activation of glucose transporters, in addition to insulin-stimulated GLUT4 translocation, play an important role in insulin-regulated glucose transport in adipocytes.