Serum Hormone and Metabolic Assays in Mice

Male C57BL/6J mice (7 weeks old) were purchased from Charles River Japan (Kanagawa, Japan) and used for the serum hormone assay and RT-PCR. Male ICR mice were purchased from Tokyo Laboratory Animals Science Co., Ltd. (Tokyo, Japan), and female PERIOD2::LUCIFERASE (PER2::LUC) knock-in mice (ICR background) bred at Waseda University were used for blood glucose measurements and in vivo imaging assays, respectively. The mice were all housed under conditions of controlled temperature (23 °C ± 3 °C), humidity (50% ± 20%), and lighting (lights on from 07:00 to 19:00 at Taisho Pharmaceutical Co., Ltd. or from 08:00 to 20:00 at Waseda University). In the 24 h cycle, Zeitgeber time 0 (ZT0) was designated as the time when the lights were turned on, and ZT12 was designated as the time when the lights were turned off. All the mice were given access to food and tap water ad libitum, and nobiletin (10–100 mg/kg; purchased from FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was administered by intraperitoneal (i.p.) injection in the vehicle (10 mL/kg body weight) containing 0.5% carboxymethyl cellulose (CMC). Rolipram (PDE4 inhibitor, FUJIFILM Wako Pure Chemical Corporation) and SR1078 (RORα/γ agonist, FUJIFILM Wako Pure Chemical Corporation) were also used for the experiment at 10 mg/kg in 0.5% CMC [12 (link)]. Mice were euthanized by rapid decapitation for whole blood samples.