Corticosterone

Blood samples were obtained from the hearts of the mice two weeks after the treadmill treatment. The plasma levels of ACTH, β-endorphin, α-melanocyte stimulating hormone (α-MSH), corticosterone, IgE, transforming growth factor (TGF)-β, histamine, tumor necrosis factor (TNF)-α, interleukin (IL)-4, IL-10, interferon (IFN)-γ, and IL-13 were determined using commercial ELISA kits (ACTH, β-endorphin and α-MSH: Phoenix Pharmaceuticals Inc., Burlingame, CA, USA; corticosterone: Assaypro, St. Charles, MO, USA; IgE: Yamasa Shoyu Co., Ltd., Chiba, Japan; TGF-β: Promega, Madison, WI, USA; histamine: Bertin Pharma, Montigny le Bretonneux, France; TNF-α, IL-4, IL-10, IFN-γ, and IL-13: R&D Systems, Minneapolis, MN, USA) according to the manufacturers’ instructions. The optical density was measured with a microplate reader (Molecular Devices, Sunny Vale, GA, USA).

All blood samples were collected in ethylenediamine-N,N,N′,N′-tetraacetic acid, dipotassium salt, and dehydrate (EDTA 2 K) containing tubes. The concentrations of the following biochemical markers were measured using commercial kits: glucose (Glu), triglyceride (TG), non-esterified fatty acid (NEFA), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) (Wako Pure Chemical Industries, Osaka, Japan); adiponectin (Otsuka Pharmaceutical, Tokyo); leptin and insulin (Morinaga Institute of Biological Science, Kanagawa, Japan); corticosterone (Assaypro, St. Charles, MO, USA); and insulin-like growth factor–1 (IGF-1) (R&D Systems, Minneapolis, MN, USA). Low-density lipoprotein cholesterol (LDL-C) was calculated as the concentration of TC, HDL-C, and TG (LDL-C = TC-HDL-C-TG/5).
For plasma amino acid analysis, plasma samples were mixed and centrifuged twice with 3% trichloroacetic acid solution. The supernatant was filtered through a 0.45 μm membrane. Plasma amino acid concentrations were measured using an automatic amino acid analyzer (L-8900; Hitachi, Tokyo, Japan). Type AN-2 and type B (Wako Pure Chemical Industries, Osaka, Japan) were used as the amino acid mixture standard solutions.
Total lipids in the liver were extracted using the Folch method, and the hepatic TG and TC concentrations were measured using commercial kits.