Six-week-old female Wistar-Imamichi rats were housed in individual cages and kept at 24±2℃ in a light-controlled room (12:12 h light:dark cycle; lights on 07:00 to 19:00) and were given free access to water and laboratory chow (Oriental MF; Oriental Yeast Co., Ltd., Tokyo, Japan). At the onset of the experiment, the rats weighed 170-180 g.

All animal procedures were approved by the Institutional Animal Care and Use Committee of Nippon Veterinary and Life Science University (Tokyo, Japan) and carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All efforts were undertaken according to the "3R principles" of the directive to reduce the number of animals used in this study and to optimize the experimental protocols to acquire the maximum amount of data from each tested animal.

At seven weeks of age, the rats were ovariectomized under sodium pentobarbital (40 mg/kg) anesthesia. Five days after surgery, the rats were divided into three groups of six rats each. The control group (group 1) was given a daily subcutaneous injection of sesame oil (0.1 mL/rat) on days 10-13 postsurgery. The remaining groups (groups 2 and 3) were injected with 2 µg of 17β-estradiol benzoate (Sigma-Aldrich, St. Louis, MO, USA) dissolved in 0.1 mL of sesame oil on days 10-12 postsurgery; half of the rats were injected with 0.5 mg of progesterone (Sigma-Aldrich) dissolved in 0.1 mL of sesame oil on day 13. Thus, the rats in group 3 (17β-estradiol benzoate+progesterone) were expected to behave like females in the proestrous stage of the estrous cycle as compared with group 2 (17β-estradiol benzoate only). Drug administration was done at 11:00 every day. All rats were anesthetized with sodium pentobarbital on day 14 postsurgery. Individual blood samples were collected from the vena cava caudalis using a syringe between 09:00 and 10:00 and centrifuged at 2,200 g for 15 min at 4℃. The serum samples were kept frozen at -35℃ until the leptin assay. Peri-uterine adipose tissue was dissected, frozen immediately in liquid nitrogen, and stored at -80℃ until RNA extraction.

Body weight and food consumption were monitored daily by weighting at 11:00; a visual check was made for any diet dropped on the floor of the cage. The food intake of each rat was measured by weighing the remaining chow.

Leptin concentrations in serum (5 µL) were determined using the Rat Leptin ELISA kit (Morinaga Institute of Biological Science Inc., Yokohama, Japan). The assay sensitivity was 200 pg/mL. The inter- and intra-assay coefficients of variation were less than 10%.

For the analysis of leptin mRNA expression, visceral abdominal fat was dissected, frozen immediately in liquid nitrogen, and stored at -80℃ until RNA extraction. Total RNA was isolated using an RNeasy Lipid Tissue Midi Kit (Qiagen, Gaithersburg, MD, USA) according to the manufacturer's instructions. The RNA samples were quantified spectrophotometrically (ND-1000 [V3.12], NanoDrop, Houston, TX, USA), and the integrity was confirmed using a 1.5% agarose gel containing 2.2 M formaldehyde. Total RNA from each sample was transcribed into first-strand cDNA using SuperScript™ III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) in a final volume of 20 µL; 1 µg of the RNA sample was mixed with 1 µL of 20 µM oligo dT Primer in DNase- and RNase-free water. The samples were then incubated at 65℃ for 10 min and placed on ice. Next, 4 µL of 5× first-stand buffer, 2 µL of 0.1M dithiothreitol, and 4 µL of 10 mM 2-deoxynucleoside 5-triphosphate mixed solution was added. The samples were preincubated at 42℃ for 2 min, and then 0.5 µL of SuperScript™ III Reverse Transcriptase was added. The samples were then incubated at 42℃ for 1 h, 30 µL of TE buffer (Tris-hydrochloride buffer, pH 8.0, containing 1.0 mM EDTA) was added, and the samples were heated at 65℃ for 10 min to inactivate the reverse transcriptase.

Real-time RT-PCR was performed using a leptin (Gene Bank Accession No.: NM_013076) specific forward primer (5'-GACCAGACCCTGGCAGTCTA-3') and reverse primer (5'-CTCAGCATTCAGGGCTAA GG-3') to amplify the product. The samples were normalized using the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Gene Bank Accession No. : NM_017008) using a forward primer (5'-TGTCAGCAATGCATCCTGCA-3') and reverse primer (5'-CCGTTCAGCTCTGGGATGAC-3'). Amplified products' size for leptin and GAPDH cDNAs are 280 bp and 240 bp, respectively. Real-time RT-PCR was performed in a final reaction volume of 25 µL using Platinum SYBR Green qPCR Supermix-UDG (Invitrogen). Each real-time RT-PCR contained 12.5 µL of Platinum SYBR Green qPCR Supermix-UDG, 10 µM each primer, and an RNA template. Real-time RT-PCR was carried out in duplicate for each sample using an ABI 7500 Real-time RT-PCR System (Applied Biosystems, Foster City, CA, USA) with the following parameters: one cycle at 50℃ for 2 min (UDG incubation), 95℃ for 2 min, and 40 cycles at 95℃ for 15 s and 60℃ for 30 s. Melting curve analyses were performed for each series to confirm the specificity of the amplified products. To confirm the absence of a genomic DNA signal, amplification without reverse transcriptase was performed. Standard curves were prepared for the tested and housekeeping genes. All expression data were normalized by dividing the amount of GAPDH used for the target gene by the amount used for the control.

All data are expressed as the mean±SEM. Statistically significant differences were evaluated by an ANOVA and Tukey's test to examine the effects among subject variables. Differences were considered significant at P<0.05.

Data related to food intake and body weight are presented in Figures 1 and 2. Notably, the food intake of the rats did not differ among the three groups before treatment, whereas groups 2 and 3 had a significantly lower food intake than group 1 following estrogen treatment. The mean food intake in groups 1, 2, and 3 was 19.51±0.51, 16.61±0.35, and 16.83±0.34 g/day, respectively, on day 12 and 19.67±0.31, 15.86±1.13, and 16.75±1.06 g/day, respectively, on day 13. The mean food intake on day 14 in group 3 (17.93±0.40 g/day) was significantly lower (P<0.05) than that in group 1 (19.99±0.46 g/day). After estrogen treatment, the rate of food intake in groups 2 and 3 decreased by 14.86% and 13.73%, respectively, compared with group 1. The mean body weights of the rats were not significantly different among the three groups.

Figure 3 shows the serum leptin levels in groups 1, 2, and 3. The serum leptin levels in group 1 (473.40±97.19 pg/mL) were significantly lower (P<0.05) than those in groups 2 and 3 (653.20±31.55 and 687.20±65.18 pg/mL, respectively). No significant difference in leptin was observed between groups 2 and 3. The expression of leptin mRNA in adipose tissue is presented in Figure 4. Leptin mRNA expression was significantly lower in group 1 (0.85±0.29; P<0.05) than in groups 2 and 3 (2.17±0.40 and 3.44±0.78, respectively). No significant differences in leptin mRNA expression were detected between groups 2 and 3. These data show that estrogen stimulates leptin mRNA expression and leptin secretion from adipose tissue.