Leptin

Blood was sampled from all patients and sera were separated from whole blood after complete coagulation, by centrifugation at 3000 rpm for 10 min. Sera were stored at −70 °C until ELISA assay analyses.
Levels of soluble RAGE, C-Terminal FGF23, Leptin, Adiponectin and Visfatin in serum were determined by commercial assays, according to the manufacturers’ instructions (sRAGE: R&D Systems, Minneapolis, Minnesota, USA; C-Terminal FGF23: Imuunotopiscs, San Clemente, CA, USA; Leptin: Enzo Life Sciences, Farmingdale, New York, USA; Adiponectin and Visfatin: AdipoGen AG, Liestal, Switzerland).
For the sRAGE assay, the sensitivity was 4.44 pg/mL, and intra- and inter-assay coefficients of variation were 2.4% and 4.7%, respectively. For the FGF23 assay, the sensitivity was 1.5 RU /mL, and intra- and inter-assay coefficients of variation were2.4% and 4.7%, respectively. According to manufacturer (, Imuunotopiscs, San Clemente, CA, USA) 1RU roughly equates to 2 pg/mL. For the Leptin assay, the sensitivity was 23.4. pg/mL, and intra- and inter-assay coefficients of variation were 4.4% and 3.7%, respectively. For the Adiponectin assay, the sensitivity was 1 ng/mL, and intra- and inter-assay coefficients of variation were 3.3% and 2.75%, respectively. For the Visfatin assay, the sensitivity was 30.0 pg/mL, and intra- and inter-assay coefficients of variation were 2.3% and 4.6%, respectively.

Freshly frozen liver tissues were homogenized and centrifuged, and the supernatants were collected [27 (link)]. The plasma and hepatic concentrations of TNF-α, IL-6, leptin, and adiponectin were then assayed (TNF-α, IL-6, leptin, and adiponectin enzyme-linked immunosorbent assay kits; all from Enzo Life Science, Farmingdale, NY, USA).

Commercially available ELISA or calorimetric kits were used to measure corticosterone, leptin, insulin, glucose, and TAG levels (corticosterone: Enzo Life Sciences, Exeter, UK; leptin and insulin: Alpha Diagnostics, Eastleigh, UK; glucose: Abcam, Cambridge, UK; TAG: Wako, Cambridge, USA). Insulin sensitivity in each group was determined by calculating QUICKI from data collected from plasma samples (1/(log [insulin (μU/L)] + log [glucose (mg/dL])) [13 (link)].
To measure plasma amino acid levels in the high fat diet groups, samples were deproteinated with 24 % (^w/_v) trichloroacetic acid at a ratio of 1:1, incubated for 10 min at room temperature, and centrifuged at 14400 rpm (Microcentaur, MSE, UK) for 10 min. Next, the supernatant was diluted with the internal standard norleucine, and the quantification of amino acids was completed using a Joel JLC-500/V amino acid analyzer (Joel Ltd., Garden City, Herts, UK) fitted with a Joel Na⁺ high performance cation exchange column.