Abstract
BackgroundOleamide (ODA) is a fatty acid primary amide first identified in the cerebrospinal fluid of sleep-deprived cats, which exerts effects on vascular and neuronal tissues, with a variety of molecular targets including cannabinoid receptors and gap junctions. It has recently been reported to exert a hypolipidemic effect in hamsters. Here, we have investigated the nuclear receptor family of peroxisome proliferator-activated receptors (PPARs) as potential targets for ODA action.ResultsActivation of PPARα, PPARβ and PPARγ was assessed using recombinant expression in Chinese hamster ovary cells with a luciferase reporter gene assay. Direct binding of ODA to the ligand binding domain of each of the three PPARs was monitored in a cell-free fluorescent ligand competition assay. A well-established assay of PPARγ activity, the differentiation of 3T3-L1 murine fibroblasts into adipocytes, was assessed using an Oil Red O uptake-based assay. ODA, at 10 and 50 μM, was able to transactivate PPARα, PPARβ and PPARγ receptors. ODA bound to the ligand binding domain of all three PPARs, although complete displacement of fluorescent ligand was only evident for PPARγ, at which an IC50 value of 38 μM was estimated. In 3T3-L1 cells, ODA, at 10 and 20 μM, induced adipogenesis.ConclusionsWe have, therefore, identified a novel site of action of ODA through PPAR nuclear receptors and shown how ODA should be considered as a weak PPARγ ligand in vitro.
Highlights
Oleamide (ODA) is a fatty acid primary amide first identified in the cerebrospinal fluid of sleepdeprived cats, which exerts effects on vascular and neuronal tissues, with a variety of molecular targets including cannabinoid receptors and gap junctions
Peroxisome proliferatoractivated receptors (PPAR) occupancy assays Since whole cell reporter gene assays may be complicated by metabolic conversion of putative ligands, we tested whether ODA was able to bind directly to PPARs, using selective ligands for each receptor as positive controls
The positive control evoked concentration-dependent inhibition of fluorescent ligand binding to the ligand binding domains of PPARα, PPARβ and PPARγ (Figure 2)
Summary
Oleamide (ODA) is a fatty acid primary amide first identified in the cerebrospinal fluid of sleepdeprived cats, which exerts effects on vascular and neuronal tissues, with a variety of molecular targets including cannabinoid receptors and gap junctions. Oleamide (ODA, (Z)-octadec-9-enamide) is a fatty acid primary amide first identified as an endogenous lipoamide in the cerebrospinal fluid of sleep-deprived cats [1]. In vivo administration of ODA has a variety of observable effects: as a sleep-inducing factor [6], eliciting hypothermia, analgesia and hypo-locomotion [7]. ODA can inhibit gap junction formation [10], modulate GABA [11] and 5-HT [12] receptors and bind to CB1 cannabinoid receptors [13]
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