Figure 5.

TNF-α inhibits the ability of IGF-I to activate IRS-2-precipitable PI3-kinase activity and neuronal survival. (A) Representative autoradiogram of a thin layer chromatogram used to measure the amount of IRS-2-precipitable PI3-kinase enzymatic activity. Cerebellar granule neurons were treated as described in the text, and PI3-kinase activity was measured in cell lysates that were precipitated with an anti-IRS-2 antibody. This autoradiogram shows that activation of IRS-2-precipitable PI3-kinase by IGF-I is inhibited by pretreatment with TNF-α. (B) TNF-α inhibits the ability of IGF-I to activate IRS-2-associated PI3-kinase activity. Results of three independent experiments show that TNF-α consistently reduces IGF-I activation of PI3-kinase from 5.1 ± 1-fold to 2.7 ± 0.7-fold (P < 0.005; n = 3). TNF-α alone does not affect lipid phosphorylation. (C) TNF-α causes neuronal degeneration by blocking the ability of IGF-I to promote neuronal survival. Cerebellar granule neurons were treated with TNF-α in the presence or absence of IGF-I (100 ng/ml). Cell survival was measured 24 h later as described in Fig. 1 (n = 3). The survival-promoting ability of IGF-I was inhibited (∗, P < 0.05) by 50% with as little as 10 pg/ml TNF-α and almost fully blocked at a concentration of 100 pg/ml. At the highest concentrations of 1,000 and 10,000 pg/ml, TNF-α alone caused a moderate reduction (P < 0.01) in neuronal survival (6% ± 3% and 5% ± 2%, respectively) as compared with cerebellar granule neurons cultured in medium alone (13% ± 3%).