We have been investigating the role of tumor necrosis factor-alpha (TNF) on the induction and regulation of the catabolic state in 3T3-Ll fibroblasts. Our interest has been focused on the regulation of cellular nutrition at the critical step of glucose entry into the cell. Our data supports the hypothesis that TNF, reminiscent of serum derived growth factors, elevates glucose transport activity as well as transporter (GLUT1) mRNA in 3T3-L1 cells by combined effects on intrinsic GLUT activity, translocation of preexisting transporters as well as GLUT1 mRNA stabilization. The GLUT1 mRNA stabilization results in a major accumulation of GLUT1 mRNA in the absence of increased transcription. The increased content of GLUT1 mRNA fuels synthesis and results in the observed major increase in glucose entry into the cell. We propose to examine mechanisms of TNF-induced stability of GLUT1 mRNA. Our studies focus on the extensive 3'-untranslated region (UTR) of the GLUT1 mRNA which contains a series of sequence motifs that may play a role in determining stability. Our preliminary data demonstrates that TNF induces the activity of a protein that binds to the 3' UTR region of the GLUT1 mRNA, which may effect the stability of the message. We propose to purify and characterize the 3'UTR binding protein(s); determine its requirements for binding and contribution to mRNA stability in a cell-free assay system. In the intact cells, using antibody prepared against the purified protein, we will characterize the synthesis, turnover and activation of the protein in response to TNF. The described studies will be performed in the 3T3-L1 preadipocytes/ fibroblasts and thoroughly characterize the regulation of GLUT1 mRNA stability. As these cells differentiate in culture to adipocytes and express a second transporter, GLUT4 (insulin responsive glucose transporter) we will examine for differential effect on the stability of both transporters. Our studies address the ability of TNF to interfere with cellular nutrition and glucose homeostasis in numerous disease states at the critical level of glucose entry into the cell.
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