These data suggest that activation of Raf-1 mediates the chronic effect of insulin on hexose uptake but is not sufficient for the rapid translocation of GLUT4. In contrast, activated Raf-1 affects neither the expression of the “insulin-responsive” glucose transporter (GLUT4) nor its cellular distribution GLUT4 is virtually undetectable on the plasma membrane in the absence of insulin and translocates normally following the addition of hormone. Total cellular GLUT1 protein is increased about 5-fold. As determined by the plasma membrane “sheet” assay, Raf-1-expressing adipocytes contain greatly elevated levels of the ubiquitous glucose transporter (GLUT1) on the cell surface in the absence or presence of insulin. Basal 2-deoxyglucose uptake in adipocytes expressing activated Raf-1 is approximately 40-fold higher than in parental adipocytes, and insulin further increases uptake about 1.2-fold. Expression of activated Raf-1 in differentiated 3T3-L1 adipocytes markedly increases hexose uptake compared to control adipocytes or those infected with the retroviral vector. To investigate a role for the Raf-1 protein kinase in insulin-stimulated glucose transport, a gene encoding an oncogenically activated Raf-1 mutant was introduced into 3T3-L1 fibroblasts by retroviral gene transfer.
Downstream mediators of insulin signaling are thought to include multiple cytoplasmic serine/threonine kinases such as the product of the cellular proto-oncogene c-raf-1.