(a) Nucleic acid extraction. Total RNA was extracted using a Power SYBR Green Cells-to-Ct kit (Catalog 4402953, Invitrogen). Briefly, frozen tissues were homogenized in ice-cold dissociating reagent in a TissueLyser (Qiagen, Germantown, MD, USA) in order to disintegrate the tissue to release nucleic acids. (b) RNA integrity check. Total RNA was resolved by electrophoresis in a 1.5% agarose gel in 1× borate solution at 100 volts for 50 min. (c) Retrotranscription for cDNA synthesis. Retrotranscription was carried out using the RT Master Mix included in the kit, under the following conditions: incubation at 37 °C for 60 min, then 95 °C for 5 min for inactivation of the RT enzyme. (d) Real time PCR. The analysis was carried out using the LightCycler 480 Instrument II real-time PCR equipment (Roche, Basel, Switzerland) under the following amplification conditions. Enzyme activation: 1 cycle at 95 °C for 10 min, PCR: 55 cycles at 95 °C for 15 s, 60 °C 1 min. Dissociation curve: default configuration of the equipment, finally 1 cooling cycle at 4 °C indefinitely. After Real-time PCR reaction, quantification cycle (Cq) was determined for each sample from the amplification plot. ∆∆Cq value was calculated by subtraction of the 36b4 Cq from each sample Cq and used for data analysis. PCR primers for SREBP1, PPARδ, AMPK, PPARγ2, adiponectin and leptin were synthesized by Sigma-Aldrich. Primers were designed using Primer-BLAST (NCBI, NIH) (Table 2).