Figure 7.

mTOR Expression, Ribosomal S6K1 Activation, and Metabolomic Changes during the Preweaning-to-Adult Transition in AhR−/− Liver

(A) Total protein obtained from preweaning (PW) and adult (AD) AhR+/+ and AhR−/− liver were analyzed for the expression of mTOR by immunoblotting using a specific antibody.

(B–D) (B) Protein extracts from the same mice were used to determine by immunoblotting the level of activation of mTORC1 target protein phospho-S6K1 (p-S6K1^Tyr389). Serum samples were obtained from preweaning (PW) and adult (AD) AhR+/+ and AhR−/− mice, processed, and their metabolites analyzed by chromatographic separation and mass spectrometry. The levels of mTORC1-activating amino acids L-Leu (C) and L-Gln (D) were measured and quantified in both AhR genotypes and developmental stages.

(E) Metabolomics were also used to identify and quantify the accumulation of intermediates of the mitochondrial oxidative metabolism succinate, fumarate, and malate in the samples indicated above.

(F) Levels of azelaic acid were determined by chromatography and mass spectrometry in serum samples from preweaning AhR+/+ and AhR−/− mice.

(G) Protein levels of the hepatic carboxylesterase-3 (CES3) were determined in preweaning AhR+/+ and AhR−/− mice by immunoblotting using a specific antibody. β-Actin was used to confirm protein integrity and equal loading.

Two representative mice for each experimental condition are shown. Eight mice (A–D) or six mice (E–F) for each developmental time and genotype and three technical replicates were analyzed. Data are shown as mean ± SD. Antibodies used are indicated in Table S1. SD, standard deviation.