Modulation of peroxisome proliferator-activated receptors (PPARs) by PPARα- and PPARγ-specific ligands and by 17β-estradiol in isolated zebrafish hepatocytes

Abstract

Peroxisome proliferation is a phenomenon occurring when responsive animals are exposed to certain compounds so-called peroxisome proliferators and is regulated through a nuclear receptor named peroxisome proliferator-activated receptor (PPAR). PPAR family members exhibit a strong binding affinity for both saturated and unsaturated fatty acids. Activators of PPARα include a variety of endogenously present fatty acids, leukotrienes and hydroxyeicosatetraenoic acids (HETEs) and clinically used drugs, such as fibrates. PPARβ activators include fatty acids, prostaglandin A 2 (PGA 2) and prostacyclin (PGI 2). PPARγ is the most selective receptor and, among others, 15-deoxy-Δ 12,14 prostaglandin J 2 (PGJ 2) has been described to be a PPARγ-specific ligand. The aim of the present study was to determine if known PPARα and PPARγ ligands were able to alter the expression of these subtypes in an in vitro model of zebrafish primary hepatocyte culture. With this purpose, a PPARα specific ligand (8S-HETE), a PPARγ specific ligand (PGJ 2) and a peroxisome proliferator of the fibrate class (clofibrate) were selected. In addition, the female hormone 17β-estradiol was also used as it is known to interact with PPARs. After cell exposure for 24 h, cells were immunohistochemically stained for both PPARs and immunolabeling was quantified as percentage of positive nuclei and cells. Levels of expression of PPARs were also measured by image analysis as grey level per cell. Expression was induced for both PPARα and PPARγ by clofibrate (at 0.5 mM for PPARα and at 1 and 2 mM for PPARγ), by HETE (1 μM), and by PGJ 2 (0.3 and 1 μM for PPARα and 0.3 μM for PPARγ). Expression of PPARγ was also induced at 10 μM by 17β-estradiol. The percentage of PPARα positive nuclei increased significantly at 1 μM HETE and the percentage of PPARγ positive cells decreased at 10 μM 17β-estradiol. As a conclusion, clofibrate, HETE and PGJ 2 are able to induce expression of both PPARα and PPARγ in zebrafish primary hepatocyte cultures. Further studies are needed to identify how the expression of different PPAR subtypes is regulated and to elucidate the implication of PPAR subtypes in zebrafish cell functions.