Supplementary methods. Materials and methods.


Figure S1 Chronic ethanol feeding and myriocin treatment effects on relative levels of phosphorylated insulin, insulin growth factor (IGF), and insulin receptor substrate (IRS) signaling molecules in liver. Liver tissue from control and chronic ethanol-fed rats that were treated with vehicle (Veh) or myriocin (Myr) was used to assess relative activation of insulin/IGF-1/IRS upstream and downstream signaling networks. Graphs depict the calculated ratios of (A) pY1162/Y1163/total insulin receptor (R), (B) pY1135/Y1136/total IGF-1 receptor, (C) pS312/total IRS-1, (D) pT202/Y204/total extracellular signal-regulated kinases 1 and 2, (E) pS473/total Akt, (F) pS9/total glycogen synthase kinase 3β, (G) pT246/total proline-rich Akt substrate 40 kDa, and (H) pT246/S112/total p70S6 kinase measured by multiplex or single-plex ELISAs. Box plots depict medians (horizontal bars), 95% confidence intervals (upper and lower limits of boxes), and ranges (stems). Intergroup comparisons were made by repeated-measures one-way anova with post hoc Tukey tests of significance.


Figure S2 Myriocin increases hepatic aspartyl-asparaginyl-β-hydroxylase (AAH) expression in chronic ethanol-fed rats. Adult male Long–Evans rats were fed with isocaloric liquid diets containing 0% (control) or 37% ethanol (caloric content) for 8 weeks and were treated with vehicle or myriocin 3 times per week over the last 5 weeks of the experiment. Western blot analysis was performed with 30 μg liver protein homogenate per lane (measured using the bicinchoninic acid assay). Blots were probed with monoclonal antibodies to AAH (A85G6) or AAH/Humbug (A85E6). The latter recognizes the full-length AAH, Humbug, and N-terminal cleavage fragments of AAH. Immunoreactivity was revealed with horseradish peroxidase-conjugated secondary antibody, enhanced-chemiluminescence reagents, and film autoradiography. Blots were reprobed with rabbit polyclonal antibodies to the p85 subunit of phosphoinositide 3-kinase as a loading control. In A and B, the upper bands represent AAH, which migrate higher than the predicted ∼86 kD due to posttranslational modification by phosphorylation. The lower bands in B represent cleavage products of AAH or Humbug. Note sharply increased levels of AAH in myriocin-treated ethanol-exposed livers. Effects in control livers are subtle, corresponding to the already lower levels of ceramide in liver.


Table S1 Effects of ethanol and myriocin on hepatic lipids*.

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