Constitutively active mitogen-activated protein kinase kinase increases GLUT1 expression and recruits both GLUT1 and GLUT4 at the cell surface in 3T3-L1 adipocytes

To address a role of mitogen-activated protein kinase (MAPK) in the regulat ion of glucose transport, we made a constitutively active mutant of MAPK ki nase (MAPKK) and introduced it into 3T3-L1 preadipocytes by using a retrovi rus-mediated transfection procedure. The deletion of 20 amino acids (those between and including 32 and 51) in the amino terminal region of Xenopus MA PKK and the replacement of serine residues on the 218 and 222 positions by glutamic acid (dSESE-MAPKK) let Xenopus MAPKK constitutively active. The is olated cell clones differently expressing dSESE-MAPKK (clone 219 higher exp ression, clone 233 lower expression) efficiently differentiated to adipocyt es by a standard differentiation cocktail. Accordingly, the increased expre ssion of dSESE-MAPKK protein during differentiation resulted in the increas ed basal MAPK activity in clone 219 adipocytes and, to a lesser extent, in clone 233 adipocytes. In contrast to clone 233 and parental adipocytes, bas al 2-deoxyglucose uptake was enhanced fourfold in clone 219 adipocytes, in accordance with increased expression of GLUT1 mRNA and protein. Whereas GLU T4 mRNA was similarly expressed in all of the adipocytes, GLUT4 protein app eared to decrease in clone 219 adipocytes. More importantly, subcellular fr actionation studies showed that the localization of both GLUT1 and GLUT4 in the plasma membranes (PMs) was markedly increased in the basal state in cl one 219 adipocytes compared with that in clone 233 and parental adipocytes, in which both glucose transporters were preferentially located in intracel lular compartments. Consequently, insulin-induced translocation of GLUT1 wa s abolished in clone 219 adipocytes, although the remaining intracellular G LUT4 was still responsive to insulin stimulation, which led to the movement to the PM. As combined effects on the situation of GLUT1 and GLUT4, the fo ldness of insulin stimulation of glucose transport based on the basal activ ity was reduced in cells expressing constitutively active MAPKK. These resu lts imply that chronic activation of MAPK could be one of the mechanisms fo r insulin resistance.