Stimulation of glucose transport and glucose transporter phosphorylation by okadaic acid in rat adipocytes.

Okadaic acid, an inhibitor of Type I and IIa protein phosphatases, was recently found to stimulate 2-deoxyglucose uptake in rat adipocytes (Haystead, T. A. J., Sim, A. T. R., Carling, D., Honnor, R. C., Tsukitani, Y., Cohen, P., and Hardie, D. G. (1989) Nature 337, 78-81). In the present experiments the effect of okadaic acid on the phosphorylation and subcellular distribution of the insulin-regulatable glucose transporter (IRGT) was investigated. At maximally effective concentrations, insulin and okadaic acid increased the amount of IRGT in the plasma membrane by 10- and 4-fold, respectively. Thus, the stimulation of glucose transport by okadaic acid was apparently due to an increase in the surface concentration of the IRGT. However, despite its stimulatory actions, okadaic acid partially inhibited the ability of insulin to enhance glucose transport and translocation of the transporter. When cells were incubated with okadaic acid alone or in combination with insulin, phosphorylation of the IRGT in the plasma membrane was increased by approximately 3-fold relative to the intracellular pool of transporters in control cells. Phosphorylation of the IRGT was confined to the presumed cytoplasmic domain at the COOH terminus of the protein. Glucose transporters were dephosphorylated in vitro by Type I or Type IIa protein phosphatases, indicating that inhibition of one or both of these phosphatases could account for the increased phosphorylation produced by okadaic acid. The observation that okadaic acid stimulated translocation of the IRGT implicated a serine/threonine phosphorylation event in triggering movement of the intracellular IRGT-containing vesicles (GTV) to the cell surface. Immunoadsorption of GTV from 32P-labeled adipocytes revealed that the IRGT was the major phosphoprotein in these vesicles. The phosphorylation of at least three other GTV proteins was increased by okadaic acid, and these species would appear to be candidates for regulators of GTV movement to the plasma membrane. It is unlikely that phosphorylation of the IRGT is the signal for translocation because insulin did not increase phosphorylation of the protein. Rather, the inhibitory effect of okadaic acid on insulin-stimulated translocation is consistent with the hypothesis that phosphorylation of the IRGT promotes its internalization.