Hyperinsulinemic-euglycemic clamp experiments

Hyperinsulinemic-euglycemic clamp experiment was performed as previously described (10). In brief, after fasting for 12 h, 5 rats in each group were anesthetized intraperitoneally (ip) with 1% pentobarbital sodium (wt/wt), followed by catheterization of the left carotid artery and internal jugular vein on both sides. Both sides of the jugular vein catheter were injected with saline, while the left carotid artery catheter was injected with heparin. After 30 min, 1 mL carotid artery blood was collected for the measurement of instant blood glucose with strip-operated blood glucose sensor (Accuchek; Roche, Germany), and basal insulin level. The left jugular vein catheter was connected to a syringe containing human insulin (Novolin R; Novo Nordisk, China), while the right jugular vein catheter was linked to infusion bags containing 10% glucose solution. The syringes and infusion bags were then connected to the two syringe pumps (Syringe pump; Terumo Holding; Japan) to make a continuous intravenous infusion. After continuous insulin infusion at a rate of 1.67 mU·kg^-1·min^-1 for 5 min, the instant blood glucose from the carotid artery was measured with strip-operated blood glucose sensor. When the values were above basal values within ±0.5 mmol/L, infusion of glucose began at a rate of 4-6 mg·kg^-1·min^-1. Blood glucose was measured every 5 min. When three continuous glucose values were within that range, insulin levels were measured again. During the whole procedure, blood glucose was measured 24 times. At the end, coefficient of variation of blood glucose (CVBG) was calculated.