Glucagon

ELISA was used to measure blood levels of a series of hormones using a series of specific commercial kits. ELISA experiments were outsourced to the ACTSI/Emory Cardiovascular Specialty Laboratories (Atlanta, Georgia). Briefly, serum samples were collected from transgenic lines (488 and 519) and their non-transgenic littermates (controls). Insulin and Glucagon were measured by ELISA using the commercial kits. The sources of commercial ELISA kits are as follows: Insulin (Mercodia), Glucagon (R&D Systems), triiodothyronine (T3) (Cal Biotech), total thyroxine (T4) (Cal BioTech), thyroid-stimulating hormone (TSH) (ALPCO), Leptin (Crystal Chem), Adiponectin (R&D Systems), TNFα (R&D Systems), IL6 (R&D Systems), and Complement C3 (Kamiya Biomedical).

The experiments were approved by the Ethical Committee for Animal Experiments of the Osaka University Graduate School of Medicine. All the in vivo experiments were performed in compliance with Osaka University’s Animal Facility regulations. We used mice of the same body weight for the group comparisons. Serum insulin (Morinaga, Japan.), glucagon (R&D, USA.) and cortisol (Cayman Chemical Company, USA.) were quantified using ELISA.

After 54 days of treatments, all of the mice were fasted for 4 hr, with water provided ad libitum, followed by collection of whole blood from the orbital cavity for determination of glucagon (R&D, Minneapolis, MN, USA), tumor necrosis factor (TNF‐α), and interleukin‐1β (IL‐1β) (R&D, Minneapolis, MN, USA).