Fluorometric assays

Before the end of the study, at weeks six and twelve, the mice were fasted for six hours in the morning. Under isoflurane anesthesia, blood was collected via cardiac punction, and euthanasia was performed by cervical dislocation. Blood samples were incubated for 30 min at room temperature and centrifuged for 15 min at 1500× g, and the obtained serum was immediately frozen and stored at −80 °C. Serum cholesterol and triglyceride levels as well as alanine aminotransferase (ALT) activity were measured by fluorometric assays (Abcam, Cambridge, UK). Serum insulin (Mercodia Ltd., Uppsala, Sweden), serum amyloid A (SAA), leptin, resistin, adipsin, and adiponectin (R&D Systems Europe Ltd., Abingdon, UK) were measured by enzyme-linked immunoassays (ELISAs). The detection limits were 33 pmol/L for insulin, 62.5 ng/L for SAA, 7.8 ng/L for leptin and resistin, 375 ng/L for adipsin, and 15.6 ng/L for adiponectin.

At the end of the study, mice were fasted for six hours (morning fast), and blood was collected under anesthesia by cardiac puncture; thereafter, the mice were euthanized by cervical dislocation. Blood samples were centrifuged for 15 min at 1500× g after 30 min incubation at room temperature, and the serum was immediately stored at −80 °C. Serum total cholesterol and triglyceride levels and alanine aminotransferase (ALT) activity were measured by fluorometric assays (Abcam, Cambridge, UK). Enzyme-linked immunoassays were used to measure the concentrations of serum amyloid A, leptin, resistin, adipsin and adiponectin (R & D Systems Europe Ltd., Abingdon, UK), and insulin (Mercodia Ltd., Uppsala, Sweden) in the serum samples. Detection limits were 62.5 pg/mL for serum amyloid A, 7.8 pg/mL for leptin and resistin, 375 pg/mL for adipsin, 15.6 pg/mL for adiponectin, and 33 pmol/L for insulin.

Six-hour fasting (morning fast) blood glucose levels in mice were measured from the tip of the tail with Contour Next One (Oy Diabet Ab, Lemu, Finland). Blood collected by cardiac puncture was centrifuged for 15 minutes at 1500 x g after 30 minutes incubation at room temperature, and obtained serum was immediately storaged at -80 °C. Serum triglyceride and total cholesterol levels, and alanine aminotransferase (ALT) activity were measured by fluorometric assays (Abcam, Cambridge, UK). Enzyme-linked immunoassays were used to measure the concentrations of leptin, resistin and adiponectin (R&D Systems Europe Ltd., Abingdon, UK), insulin (Mercodia Ltd., Uppsala, Sweden) and serum amyloid A (Tridelta Development Ltd., Maynooth, Ireland) in serum samples. Detection limits were 7.8 pg/mL for leptin and resistin, 15.6 pg/mL for adiponectin, 33 pmol/L for insulin and 16 ng/mL for SAA.