Tumor necrosis factor-alpha- and hyperglycemia-induced insulin resistance - Evidence for different mechanisms and different effects on insulin signaling

Inhibition of insulin receptor signaling by high glucose levels and by TNF-alpha was recently observed in different cell systems. The aim of the present study was to characterize the mechanism of TNF-alpha-induced insulin receptor inhibition and to compare the consequences of TNF-alpha- and hyperglycemia-induced insulin receptor inhibition for signal transduction downstream from the IR. TNF-alpha (0.5-10 nM) and high glucose (25 mM) showed similar rapid kinetics of inhibition (5-10 min, > 50%) of insulin receptor autophosphorylation in NIH3T3 cells overexpressing the human insulin receptor. TNF-alpha effects were completely prevented by the phosphotyrosine phosphatase (PTPase) inhibitors orthovanadate (40 mu M) and phenylarsenoxide (35 mu M), but they were unaffected by the protein kinase C (PKC) inhibitor H7 (0.1 mM), the phosphatidylinositol-3 kinase inhibitor wortmannin (5 mu M), and the thiazolidindione troglitazone (CS045) (2 mu g/ml). In contrast, glucose effects were prevented by PKC inhibitors and CS045 but unaffected by PTPase inhibitors and wortmannin. To assess effects on downstream signaling, tyrosine phosphorylation of the following substrate proteins of the insulin recepter was determined: insulin receptor substrate-1, the coupling protein She, focal adhesion kinase (FAK(125)), and unidentified proteins of 130 kD, 60 kD, and 44 kD. Hyperglycemia (25 mM glucose) and TNF-alpha showed analogous (> 50% inhibition) effects on tyrosine phosphorylation of insulin receptor substrate-1, She, p60, and p44, whereas opposite effects were observed for tyrosine phosphorylation of FAK(125), which is dephosphorylated after insulin stimulation. Whereas TNF-alpha did not prevent insulin-induced dephosphorylation of FAK(125), 25 mM glucose blocked this insulin effect completely. In summary, the data suggest that TNF-alpha and high glucose modulate insulin receptor-signaling through different mechanisms: (a) TNF-alpha modulates insulin receptor signals by PTPase activation, whereas glucose acts through activation of PKC. (b) Differences in modulation of the insulin receptor signaling cascade are found with TNF-alpha and high glucose: Hyperglycemia-induced insulin receptor inhibition blocks both insulin receptor-dependent tyrosine, phosphorylation and dephosphorylation of insulin receptor substrate proteins. In contrast, TNF-alpha blocks only substrate phosphorylation, and it does not block insulin-induced substrate dephosphorylation. The different effects on FAK(125) regulation allow the speculation that long-term cell effects related to FAK(125) activity might develop in a different way in hyperglycemia- and TNF-alpha-dependent insulin resistance.