Serum Ghrelin and Lactate Measurement Protocol

At sacrifice, 2–3 h after lights on, the serum was used for measurement of ghrelin and lactate, using a specific enzyme-linked immunosorbent assay kit^{15 (link)–20 (link)}. The low and high detection limits for ghrelin were 0.04 and 10 ng/mL (kit number EZRGRA-90K; Merck Millipore, Rosh Haayin, Israel), and the intra- and inter-assay CVs were 1.1% and 3.2%, respectively. Serum lactate was determined at the Soroka University Medical Center Biochemistry laboratory. In a subset of n = 4 of control animals, AO, and OR arterial blood gases (pH, PCO₂, PO₂, and HCO₃⁻) were determined in parallel to their serum lactate level on day 66 after surgery. Antibodies used for evaluation of the hypothalamic protein extract by western immunoblot analysis (see Supplementary Methods) were GHSR1a (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Akt and p-Akt (Cell Signaling Technology, Danvers, MA, USA), soleus AMPK and p-AMPK (MP Biomedical Solon, OH, USA), and GAPDH (Proteintech, Rosemont, IL).