A role for Raf-1 in the divergent signaling pathways mediating insulin-stimulated glucose transport.
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. 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. 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. 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. 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. Total cellular GLUT1 protein is increased about 5-fold. 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. 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. Moreover, the differential effects of activated Raf-1 expression on the two transporter isoforms define divergent signaling pathways by which insulin regulates glucose transport in cultured adipocytes.
Fingar
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Program in Cell and Developmental Biology, Harvard Medical School, Boston, Massachusetts 02115.
Birnbaum
M J
MJ
D016475:3T3 Cells; D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003847:Deoxyglucose / Q000378:metabolism*; D004789:Enzyme Activation; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism*; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis* / Q000378:metabolism; D009124:Muscle Proteins; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D011519:Proto-Oncogenes; D015398:Signal Transduction; D014162:Transfection
D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D011518:Proto-Oncogene Proteins; C495678:Slc2a1 protein, mouse; C495693:Slc2a4 protein, mouse; D003847:Deoxyglucose; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D005947:Glucose