Ec3001 1

Trunk blood samples were collected into tubes containing 5 μl heparin. Then, the samples were centrifuged at 830 g for 15 min at 4°C to separate the plasma, which was assayed using a commercially available CORT ELISA kit per the manufacturer's instructions (EC3001‐1, AssayPro, St. Charles, MO, USA).

Hemospasia from the heart was done at the 4th, 6th and 8th week of feeding at 2 to 4 PM, when the plasma levels were similar to the 24-h average level [18 (link)]. After blood sampling, the rats were fasting for 16–18 h, then hemospasia was executed again to collect the fasting blood samples. The samples were centrifuged at 3000 g for 10 min and the supernatant as plasma was taken. Plasma triglyceride, cholesterol, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentrations were measured by an analyzer (Cobas C501, Roche Ltd., Basel, Switzerland); plasma insulin, aldosterone and corticosterone concentrations were analyzed by ELISA (10-1250-01, Mercodia AB, Uppsala, Sweden; EC3001-1, Assaypro LLC, St Charles, MO, USA; 10004377, Cayman Chemical, Ann Arbor, MI, USA). Plasma glucose concentrations were determined by a glucose meter (AlphaTRAK, Abbott, Chicago, IL, USA). The blood glucose, triglyceride, cholesterol and insulin levels were analyzed from the fasting blood samples and ALT, AST, CRH, ACTH, aldosterone and corticosterone levels were analyzed from the non-fasting blood samples at 2 to 4 PM. HOMA-IR (Homeostatic Model Assessment for Insulin Resistance), the index of insulin resistance, was calculated as insulin (µU/mL) × glucose (mg/L)/22.5.