Rat insulin radioimmunoassay

INS-1 cells were pre-incubated with myricetin (20 µM) for 1 hour (5% CO₂, 37℃) in RPMI medium and washed twice in Krebs-Ringer bicarbonate buffer (114 mmol/L NaCl, 4.4 mmol/L KCl, 1.28 mmol/L CaCl₂, 1 mmol/L MgSO₄, 29.5 mmol/L NaHCO₃, 10 mmol/L HEPES, 2.8 mmol/L glucose, and 0.1% bovine serum albumin, pH 7.4). After that cells were incubated for 1 hour in krebs ringer bicarbonate buffer with the basal (2.8 mmol/L) or the stimulatory (16.6 mmol/L) glucose with or without myricetin. The supernatant was carefully collected and subjected to rat insulin radioimmunoassay (Linco Research, St. Charles, MO, USA).

To evaluate the dose‐dependent antidiabetic effect of GLP‐1‐gFc in the progress of type 2 diabetes, 5‐week‐old male diabetic (C57BL/KSJ‐db/db) mice were acclimated to a feeding environment for 2 weeks. After evaluation of body weight, animals were allocated to treatment groups (n = 12/group): vehicle, 0.6 mg/kg dulaglutide, 0.6 mg/kg GLP‐1‐gFc and 1.8 mg/kg GLP‐1‐gFc. The TAs were administered subcutaneously twice a week for 4 weeks. Non‐fasting blood glucose and insulin were measured biweekly, and overnight fasting blood glucose, insulin and HbA1c were measured at sacrifice. Glucose was analysed using GM9 Glucose Analyser (Analox Instruments Ltd, UK). Insulin and HbA1c were measured by rat insulin radioimmunoassay (LINCO Research, MO, USA) and Vantage analyser (Siemens, Munich, Germany), respectively.