Figure 3. Effects of aging and PASK deficiency on the expression of several hepatic genes involved in mitochondrial biogenesis and PGC1a and SIRT1 proteins. Real-time PCR was used to analyze the expression of Nrf2 (A), Pparg (B), Ppargc1a (C) and Sirt1 (D) genes. The mRNA levels were measured in the livers of non-fasted (NON-FASTED) and 24-h fasted (FASTED) young (3-5 months) and aged (18-20 months) wild-type (WT) and PASK-deficient (Pask-/-) mice. Immunoblot analysis of PGC1a (E) and SIRT1 (F) in livers from young and aged WT and Pask-/- mice. Liver lysates from non-fasted (NF) and 24-h fasted (F) mice were processed for western blot analysis. The value obtained in 3-5-month-old non-fasted WT mice was taken as 1. Bar graphs in (A–D) represent the means ± SEM, and the levels of expression were normalized by mRNA of β-actin used as housekeeping gene;(E, F) represent the means ± SEM of the densitometric values normalized by total protein detected by Stain-Free (TOTAL PROTEIN) (Supplementary Figure 1); n = 4-5 animals per condition. $P < 0.05, $$P < 0.01, $$$P < 0.001 3-5 months vs. 18-20 months; * P < 0.05, ** P < 0.01, *** P < 0.001 WT vs. Pask-/-; #P < 0.05, ##P < 0.01, ###P < 0.001 non-fasted vs. fasted.