The mRNA expression of acetyl-CoA carboxylase 1 (ACC1), AMPKα1 and AMPKα2 in hepatic tissues, and mRNA expression of leptin, uncoupling protein 2 (UCP2), adiponectin, CCAAT/enhancer-binding protein (C/EBP)α, C/EBPβ and sterol-regulatory-element-binding protein 1c (SREBP1c) in periovarian adipose tissue were determined using RT-qPCR (46 (link)). Briefly, RNA from adipose tissues was isolated using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The RNA quality and quality were assessed using the CFX96TM Real-Time System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The isolated RNA samples were treated with recombinant DNase I (Ambion; Thermo Fisher Scientific, Inc.) and reverse-transcribed using a high-capacity cDNA RT kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). The analyses were performed using the ABI Step One Plus Real-Time System (Applied Biosystems; Thermo Fisher Scientific, Inc.) (47 ), with mRNA expression calculated relative to the vehicle control. The following thermal conditions were applied: 94°C for 10 min; 39 cycles of 94°C for 15 sec and 57°C for 20 sec; and 72°C for 30 sec. The data were normalized to GAPDH mRNA expression using the comparative threshold cycle method (48 (link)). The oligonucleotide primer sequences used for PCR are listed in Table I.