Clofibrate

Cell experiment:

Cells are seeded at a density of 2.5 × 104 cells/well (for WST-1, intracellular lipid droplet quantification and dichlorofluorescein (DCF) assay, 96-well plates) and 1 × 105 cells/well (for Nile Red Staining, 12-well plates) in MEM/EBSS medium and incubated overnight for adherence. The next day cell culture medium is replaced with freshly prepared medium containing the fatty acid mixture oleate:palmitate (2:1) in presence of 3% fatty-acid-free bovine serum albumin. Cells are treated with 0, 0.5, 1, 2, and 3 mM fatty acid (FA) mixture for 24 and 48 hr at 37°C in a humidified incubator in an atmosphere of 95% air and 5% CO2. Clofibrate is used to increase levels of FABP1 in treated cell cultures. Clofibrate (500 μM) is dissolved in DMSOand later added to the medium (DMSO < 0.1% v/v in final volume). Control cells are incubated with DMSO alone. Four different cell treatments includ 1-day FA treatment, 2-day FA treatment, early clofibrate intervention and late clofibrate intervention[1].

Animal experiment:

Female and male C57BL/6JNarl mice are used for breeding. Females with parity from 1 to 5 are used. Pregnant females are fed either a control (C) or experimental (CF) diet from breeding to parturition. The C diet is based on an AIN-93M diet with a slight modification to contain 21 kcal% fat from soybean oil, whereas the CF diet is the C diet with addition of 0.5% clofibrate. Pregnancy is dated by the presence of a vaginal plug (defined as pregnancy day 1). After spontaneous parturition (pregnancy day 19.5 ± 0.5), all littermates are uniformly nursed by dams fed the C diet for 3 wk, with litter sizes adjusted to 8-10, weaned onto a nonpurified standard diet for 4 wk, and then switched to a HFD (51 kcal% fat, butter-based) for 5 wk. In this study, only male offspring are used and 2 groups of offspring are designated, according to their mother's diet (C or CF). All mice are kept in a room maintained at 23 ± 2°C, with a controlled 12-h-light:-dark cycle with ad libitum to feed and drinking water. Body weight and feed intake are recorded weekly[3].

References:

[1]. Willson TM, et al. The PPARs: from orphan receptors to drug discovery. J Med Chem. 2000 Feb 24;43(4):527-50.
[2]. Chen Y, et al. Clofibrate Attenuates ROS Production by Lipid Overload in Cultured Rat Hepatoma Cells. J Pharm Pharm Sci. 2017;20(0):239-251.
[3]. Chen SH, et al. Prenatal PPARα activation by clofibrate increases subcutaneous fat browning in male C57BL/6J mice fed a high-fat diet during adulthood. PLoS One. 2017 Nov 2;12(11):e0187507.