Lactate measurement

Lactate levels were determined in hypothalamic tissues by high-performance liquid chromatography (HPLC). On the day of the experiment, tissues were homogenized with 150 μl lactate assay buffer (Abcam) and centrifuged at 4 °C at 10,000×g for 4 min. Supernatants were filtered through 10 kDa MW spin filters (Abcam) to remove all proteins. Lactate concentration was measured with an HPLC autosampler system (Shimadzu LC-20AT; Kyoto, Japan) equipped with four pumps and an SPD-M20A diode array detector. Then 20 μl of each sample was injected and the chromatographic separation was performed with an Inertsil C18 ODS-3 column (5 μm particle size, 4.6 mm × 250 mm; Japan). The analysis was isocratic at λmax: 210 nm, 65 °C (column temperature), and flow rate of 0.6 ml/min, with a mixture of water (adjusted to pH 2.0 with H₂SO₄) and acetonitrile as the mobile phase (95:5, v/v; total retention time was 30 min). The mobile phase was freshly prepared, passed through a 0.45 μm membrane filter to remove any particulate matter, and degassed by sonication before use. The sensitivity of the detector was set at 0.01 AUFS. Prior to sample injection, the column was equilibrated for at least 30 min with the mobile phase flowing through the system. Each sample was injected in triplicate with a relative standard deviation below 0.73% and 9.82% for standard samples and real samples, respectively. The lactate concentration was calculated against a standard curve using the area values of each pick.