FIGURE 11:

JNKs are not required for insulin resistance in ER-stressed cells. (A) Activation of JNK in Hep G2 cells exposed to different concentrations of thapsigargin, but not tunicamycin or SubAB, for 18–36 h. The arrows indicate the p46 and p54 isoforms of JNKs; the three lines phosphorylated species of p38, p42, and p44 MAP kinases. Bars represent SEs (n = 3). p values for comparison of treated samples to the untreated sample (“–”) were calculated by ordinary two-way ANOVA with Tukey’s multiple comparisons test. (B) JNKi VIII and XVI inhibit phosphorylation of c-Jun at S63 in Hep G2 cells exposed to the indicated concentrations of thapsigargin for 36 h. Hep G2 cells were treated with 8 μM JNKi VIII or XVI for 0.5 h before exposure to thapsigargin in the presence of 8 μM JNKi VIII or XVI or no JNK inhibitor (“–”) for 36 h. The bar graphs show S63 phosphorylation of c-Jun standardized to c-Jun levels and c-Jun levels standardized to GAPDH levels. Bars represent SEs (n = 3). p values were calculated by ordinary two-way ANOVA with Tukey’s multiple comparisons test on square root or arctangent-transformed data, respectively. (C–E) JNKi VIII and XVI do not reverse inhibition of insulin-stimulated phosphorylation of AKT at S473 by thapsigargin in Hep G2 cells. Hep G2 cells were treated for 0.5 h with 8 μM JNKi VIII or XVI, followed by exposure to the indicated concentrations of thapsigargin in the presence of 8 μM JNKi VIII or XVI or no JNK inhibitor (“–” ) for 36 h. Cells were serum-starved in the last 18 h of thapsigargin treatment and then stimulated with 10 or 100 nM insulin for 15 min in the continued presence of thapsigargin and JNK inhibitors, where applicable. (C) Western blots for pS473-AKT, AKT, and GAPDH. (D) Quantification of the Western blots in panel C. Bars represent SEs (n = 3). p values were calculated by ordinary two-way ANOVA with Dunnett’s multiple comparisons test. (E) Reanalysis of the data in panel D after normalization of data for each condition of JNK inhibition to the insulin-stimulated sample not exposed to thapsigargin for the corresponding condition. p values were calculated by ordinary two-way ANOVA with Dunnett’s multiple comparisons test. (F) JNKi VIII and XVI do not restore levels of insulin receptor β chains or restore processing of α-β proreceptors to levels of untreated cells. Hep G2 cells were treated with 8 μM JNKi VIII or XVI for 0.5 h before exposure to thapsigargin in the presence of 8 μM JNKi VIII or XVI or no JNK inhibitor (“–”) for 36 h. Bars represent SEs (n = 3 for β chains and n = 4 for α-β proreceptors). p values were calculated by ordinary two-way ANOVA with Tukey’s multiple comparisons test.