Abstract
A number of studies have demonstrated that tumor necrosis factor-alpha (TNF-alpha) is associated with profound insulin resistance in adipocytes and may also play a critical role in the insulin resistance of obesity and non-insulin-dependent diabetes mellitus. Reports on the mechanism of TNF-alpha action have been somewhat contradictory. GLUT4 down-regulation has been implicated as a possible cause of insulin resistance as has been the reduced kinase function of the insulin receptor. Here we examine the effects of tumor necrosis factor on the protein components thought to be involved in insulin-stimulated glucose transport in adipocytes, namely the insulin receptor, its major substrate IRS-1, and the insulin responsive glucose transporter GLUT4. Prolonged exposure (72-96 h) of 3T3-L1 adipocytes to TNF-alpha causes a substantial reduction (>80%) in IRS-1 and GLUT4 mRNA and protein as well as a lesser reduction (>50%) in the amount of the insulin receptor. Nevertheless, the remaining proteins appear to be biochemically indistinguishable from those in untreated adipocytes. Both the insulin receptor and IRS-1 are tyrosine-phosphorylated to the same extent in response to acute insulin stimulation following cellular TNF-alpha exposure. Furthermore, the ability of the insulin receptor to phosphorylate exogenous substrate in the test tube is also normal following its isolation from TNF-alpha-treated cells. These results are confirmed by the reduced but obvious level of insulin-dependent glucose transport and GLUT4 translocation observed in TNF-alpha-treated adipocytes. We conclude that the insulin resistance of glucose transport in 3T3-L1 adipocytes exposed to TNF-alpha for 72-96 h results from a reduced amount in requisite proteins involved in insulin action. These results are consistent with earlier studies indicating that TNF-alpha reduces the transcriptional activity of the GLUT4 gene in murine adipocytes, and reduced mRNA transcription of a number of relevant genes may be the general mechanism by which TNF-alpha causes insulin resistance in adipocytes.
Highlights
On the other hand, numerous recent studies have implicated the involvement of TNF-␣ in insulin resistance in adipocytes in culture as well as in whole-animal models
Effect of TNF-␣ on Insulin-sensitive Glucose Uptake—In hepatocytes, it has been reported that TNF-␣ induces a defect in insulin signaling in a time frame of less than 1 h [16, 17]
The recent results from several groups indicating that TNF-␣ may cause rapid [17, 18] or slow [13,14,15] alterations in the ability of insulin to phosphorylate insulin receptor substrate-1 (IRS-1) in hepatocytes [17, 18] and adipocytes [13,14,15] prompted us to re-examine this process in 3T3-L1 adipocytes
Summary
Human or murine TNF-␣ was dissolved in phosphate-buffered saline containing 0.1% fatty acid-free and growth factor-depleted bovine serum albumin (Sigma) and was added to the cell culture media 7 days after the induction of differentiation when greater than 95% of the cells had the morphological and biochemical properties of adipocytes. Western blots employing a monoclonal antibody were analyzed using goat antimouse antibody (Sigma) coupled to horseradish peroxidase followed by chemoluminescence using the Renaissance system (Dupont NEN). Exogenous Kinase Activity Assays—The exogenous kinase activity of the insulin receptor following its solubilization and partial purification from cell extracts was determined as described previously [24]. Radioactivity was normalized by the amount of the insulin receptor, which was determined by Western blot as described previously [24]. Nonspecific uptake and absorption was always less than 10% of the total uptake
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