Human Adipocyte Fatty Acid-Binding Protein (aP2) Gene Promoter-Driven Reporter Assay Discriminates Nonlipogenic Peroxisome Proliferator-Activated Receptor γ Ligands

Abstract

Peroxisome proliferator-activated receptors (PPARs) regulate storage and catabolism of fats and carbohydrates. PPAR␥ activity increases insulin sensitivity and adipocyte differentiation at the expense of adipogenesis and weight gain. The goal of this study was to 1) clone the promoter of the human adipocyte fatty acid binding protein (aP2) gene, namely fatty acid-binding protein-4, 2) characterize its pharmacological regulation, and 3) determine its putative predictability for adipogenesis. Among the selected PPAR agonists, rosiglitazone and pioglitazone displayed the highest maximal efficacy (E max ) on reporter-gene assays in COS-7 cells cotransfected by either a galactosidase 4-response elementbased or a human aP2 promoter-based Luc reporter vector, along with either chimeric or full-length human PPAR expression plas- -0072), and indomethacin behaved as partial agonists relative to pioglitazone in full-length human aP2-PPAR␥2. Beyond their partial PPAR␥ agonist properties, these compounds elicited a lower maximal up-regulation of mouse aP2 mRNA in 3T3-L1 adipocytes as compared with pioglitazone; these properties paralleled a time-dependent increase in neutral lipids. By contrast, the selective PPAR␣ agonist 2,2-dichloro-12-(4-chlorophenyl)dodecanoic acid (BM-17.0744) neither stimulated the human aP2-PPAR␣ promoter reporter-gene assay, thus demonstrating a specific interaction between PPAR␥ and the aP2 promoter, nor affected lipogenesis in 3T3-L1 cells. Altogether, these data characterized a functional promoter of the human aP2 gene; its in vitro pharmacological regulation in PPAR␥-mediated reporter-gene assay may represent an interesting complement or an alternative to time-consuming procedures aiming at discriminating PPAR ligands with low lipogenic properties.Metabolic syndrome is characterized by the clustering of at least three risk factors among hypertension, certain types of dyslipidemia, impaired glucose tolerance and type II diabetes, and obesity. These metabolic abnormalities lead to atherosclerosis and related complications (Haffner and Taegtmeyer, 2003). The control of lipid and carbohydrate metabolism, including physiologic and pharmacological treatments, represents a valid rationale to reduce cardiovascular diseases in patients with metabolic syndrome (Beckman et al., 2003;Wilson and Grundy, 2003).PPARs are a subclass of the nuclear receptor superfamily acting as ligand-dependent transcription factors (Kersten et al., 2000). Three subtypes were identified; the ␣ isoform is the primary subtype expressed in liver, but also in heart and kidney, and acts as a major regulator of metabolism of fats, catabolism of fatty acids, and synthesis and catabolism of lipoproteins (Barbier et al., 2002). PPAR␣ is also involved in Parts of this work were presented as a poster at the 74th Congress of the European Atherosclerosis Society (April 17-20, 2004, Seville, Spain).Article, publication date, and citation information can be found at

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“…FABP4 facilitates transport of fatty acid ligands to the nucleus, 35,36 where it interacts directly with PPAR␥. 37,38 Various data support the concept that FABP4 is at the crossroads of 2 central pathways in macrophages, where it coordinates cholesterol metabolism and inflammatory response. 39 In mice, it appears that FABP4 is required for expression of inflammatory cytokines and/or chemoattractants in macrophages and adipose tissue.…”

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“…Sequence analysis showed that there was no PPRE-like motif in human A-FABP promoter. 40 Rival et al 41 all the same have characterized a functional promoter of the human A-FABP gene and showed that rosiglitazone and pioglitazone displayed a high efficacy on reporter-gene assays in COS-7 cells cotransfected by a human A-FABP promoter-based Luc reporter vector and fulllength human PPAR expression plasmids. Surprisingly, in these experiments, a selective PPARa agonist (BM-17.1744) did not stimulate the human A-FABP-PPARa promoter reporter-gene assay.…”

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“…GRO-seq (Core et al 2008;Wang et al 2011) was used to measure nascent gene transcription in mouse 3T3-L1 adipocytes and after treatment with rosi for 10 min, 30 min, 1 h, and 3 h. The Fabp4 locus, a classic adipocyte PPARg gene target (Spiegelman and Green 1980;Rival et al 2004), showed increased transcription with rosi treatment (Fig. 1A).…”