(a–h) Peroxisome proliferation in liver cells of wild-type (WT), SRC-1-/- SRC-2-/- and SRC-3-/- mice treated with Wy-14,643 for 4 days. Liver sections were processed for the cytochemical localization of peroxisomal catalase by using alkaline ,-diaminobenzidine substrate. Control diet (upper panels; a, c, e, g). Wy-14,643 diet (lower panels; b, d, f, h). Peroxisomes appear as brown dots (arrows) distributed throughout the cytoplasm in these 0.5 m thick sections. All mice, wild-type and SRC nulls displayed extensive peroxisome proliferation after treatment with Wy-14, 643 indicating that these coactivators are not required for PPARα-regulated pleiotropic responses including fatty acid oxidation. (i) Northern blot analysis to confirm changes in mRNA expression of peroxisomal and microsomal fatty acid metabolizing enzymes in wild-type and SRC nulls after 4-days treatment with PPARα ligand Wy-14, 643. All genes are regulated by PPARα. Fatty acyl-CoA oxidase-1 (ACOX1), peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme (L-PBE), and peroxisomal thiolase (PTL) represent peroxisomal β-oxidation system while CYP4A1 is involved in microsomal ω-oxidation of fatty acids. GAPDH is used as an indicator of RNA loading. (j) Western blot analysis from the above-mentioned livers was used to verify the degree of expression of peroxisomal and mitochondrial fatty acid metabolizing enzymes in wild-type and SRC knockout mice. Liver homogenates (20 g) from each group of mice were run on 4–20% SDS PAGE gel and immunoblotted using antibodies for peroxisomal (ACOX1, L-PBE, PTL, D-PBE, and SCPx) and mitochondrial (SCAD, MCAD, and VLCAD) fatty acid metabolizing enzymes. No difference in the induction was observed between SRC nulls and wild-type for the β-oxidation enzymes.
