Unliganded PPARδ actively represses the basal expression of target genes. Wild-type or mutant PPARδ receptors were reintroduced into a PPARδ−/− monocytic cell line derived from bone marrow. RNAs were collected from cells treated with (+) or without (−) agonist GW1516, and the expression of target genes was analyzed by Northern blot. PPARδΔAF2, AF2 domain deletion; DBD-M, DNA-binding-deficient mutant.

PPARδ-dependent regulation of target genes in primary macrophages. Northern blot analysis of target gene expression in wild-type and PPARδ−/− peritoneal macrophages. RNAs were collected from thioglycollate-elicted peritoneal macrophages treated with vehicle (c) or ligands for PPARα [Wy14,643 (Wy), 30 μM], PPARδ (GW1516, 0.1 μM), and PPARγ [rosiglitazone (BRL), 1 μM]. V/L: VLDL (10 μg/ml) plus lipoprotein lipase (LPL, 10 μg/ml).

PPARδ activation increases fatty acid catabolism. (A and B) PPARδ ligand potentiates fatty acid β-oxidation. Macrophages were pretreated with PPAR ligands for 24 h and labeled with ³H oleate. The fatty acid uptake and accumulation rate were determined by measuring cellular unprocessed ³H oleate within 30 min (A). Solid and dotted lines represent wild-type and PPARδ−/− cells, respectively. The β-oxidation rate was determined by measuring the amount of ³H₂O in culture supernatant 4 h after loading of radiolabeled fatty acids (B). (C) PPARδ activation initiates a fat-burning program. The diagram indicates three major steps in the β-oxidation pathway, including (I) carnitine biosynthesis, (II) mitochondrial β-oxidation, and (III) peroxisomal β-oxidation. Genes regulated by PPARδ are in gray-shaded text.

TTS2 potentiates VLDL-mediated PPARδ activation. (A) Northern blot analysis of tissue distribution of the mouse TTS2 and adiponutrin gene. Actin probe serves as a loading control. (B) The time course of TTS2 and adiponutrin expression during adipocyte differentiation of 3T3-L1 cells. RNAs were collected from days 0–8 of the differentiation process. Perilipin is a marker for mature adipocytes. (C) Western blot analysis of the subcellular localization. CV-1 cells were transfected with a FLAG-tagged TTS2 or adiponutrin construct and subjected to fractionation. Cellular localization was determined by immunoblotting. Cyp450 reductase and heat-shock protein (HSP) 90 are markers for the membrane and cytosol fraction, respectively. C, cytosol; M, membrane; N, nucleus. (D) TTS2 enhances the ability of VLDL to activate PPARδ. CV-1 cells were transfected with a GAL4 reporter and a GAL4-PPARδ ligand-binding domain fusion (GAL4-PPARδLBD), in the presence or absence of TTS2 or adiponutrin. Cells were treated with VLDL at various concentrations.

Activation of PPARδ induces the expression of key genes in the fatty acid catabolic pathway. (A) Northern blot analysis of PPARδ target genes. RNAs were isolated from RAW 264.7 cells expressing either yellow fluorescent protein control or PPARδ in the absence (−) or presence (+) of a specific agonist GW501516 (GW1516). PPARδ target genes identified by DNA array were examined by Northern blot hybridization. The actin probe was included as a loading control. (B) ALDH9 is a direct target gene for PPARδ. Binding of PPARs to the putative PPRE identified from the ALDH9 promoter was examined by the gel mobility assay by using radiolabeled oligomers and in vitro translated proteins (Upper). Up to 100-fold (100×) excess of unlabeled probe was included in the reaction for the binding competition experiment. Nonspecific element (NS) and PPRE from the acyl-CoA synthetase gene was included as a negative and positive control, respectively. The 5′ regulatory region of the mouse ALDH9 gene was subsequently cloned into a luciferase reporter construct (Upper). Numbers denote the positions relative to the transcriptional initiation site. The sequence of the PPRE and its mutation is indicated. The induction of the ALDH9 promoter activity was tested in RAW cells cotransfected with PPARδ and the obligate heterodimer partner RXR. (C) PPARδ regulates TTS2 promoter activity in a PPRE-dependent manner. YFP, yellow fluorescent protein; RLU, relative luciferase unit. Concentration for GW1516, 0.1 μM; VLDL plus LPL, 10 μg/ml each.