Figure 3: Acetaldehyde does not impair insulin stimulated signaling through ERK and Akt. Rat cerebellar neuron cultures seeded into 96-well plates, were treated with 3.5 mM acetaldehyde for 48 hours, and then stimulated with 10 nM insulin for up to 60 minutes. Cells were fixed and used to measure (a) pERK, (b) pAkt, (c) total ERK, and (d) total Akt by cellular ELISA, and results were normalized to H33342 fluorescence. The ratios of (e) pERK/total ERK and (f) pAkt/total Akt were calculated to show relative time-dependent shifts in insulin-stimulated signaling. Graphs depict the mean ± S.E.M. of results generated from 8 replicate assays. These results are strikingly different from those obtained using ethanol-treated cultures (see Figure 4). Inter-group differences with respect to pERK, pAkt, ERK, and Akt are statistically significant by area-under-curve analysis. However, relative levels of ERK and Akt phosphorylation are similar for vehicle and acetaldehyde-treated cultures.