2.4. Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) Analysis

RT-qPCR was used to measure mRNA expression levels of leptin receptor (Lepr), suppressor of cytokine signalling-3 (Socs3), insulin receptor (Insr), neuropeptide Y (Npy), agouti-related peptide (Agrp), brain-derived neurotrophic factor (Bdnf), pro-opiomelanocortin (Pomc), cocaine- and amphetamine-regulated transcript (Cart), and melanocortin 4 receptor (Mc4r) in hypothalamus, and low-density lipoprotein receptor-related protein (Lrp11), glutaminase (Gls), uncoupling protein 2 (Ucp2), and NPC intracellular cholesterol transporter 1 (Npc1) in PBMC. For hypothalamus, 0.25 µg of RNA (in a final volume of 12.5 µL) were denatured at 65 °C for 10 min and reverse transcribed using MuLV reverse transcriptase (Applied Biosystems, Madrid, Spain) at 25 °C for 10 min, 37 °C for 50 min, and 70 °C for 15 min. In the case of RNA from PBMC, cDNA was obtained using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, SA, Madrid, Spain), as previously described [31]. RT-qPCR reactions were carried out from diluted cDNA template (1/10 for hypothalamus and 1/5 for PBMC), forward and reverse primers (10 μM), and Power SYBER Green PCR Master Mix (Applied Biosystems, CA, USA), using the Applied Biosystems StepOne Plus Real-time PCR Systems (Applied Biosystem). The temperature profile of the reactions was 10 min at 95 °C and 40 two-temperature cycles of 15 s at 95 °C and 1 min at 60 °C, and a melting curve was also obtained after each run to confirm the purity of the products. The threshold cycle was determined by the StepOne Software v2.2.2, and the relative expression of the analyzed genes was expressed as a percentage of CON-V male animals and using the 2^−ΔΔCt method. Reference genes used in this analysis were β-actin and guanosine diphosphate dissociation inhibitor 1 (Gdi1). Sequences and amplicon size of primers (Sigma, Madrid, Spain) are detailed in Supplementary Material Table S1.