One touch ultra 1

Glucose tolerance test was performed in overnight fasted mice. Blood samples were taken from the tail to measure blood glucose at baseline before mice were injected with glucose (2 mg/kg body weight, IP, Sigma-Aldrich). To test for the glucose-reducing effect of insulin, mice were fasted for 5 h. After determining baseline glucose levels, mice were injected with insulin (0.5 unit/kg body weight, IP, Novo Nordisk). Blood glucose was measured at 15, 30, 60, and 120 min after injection of glucose or insulin. Blood glucose levels were determined using a glucometer (OneTouch Ultra 1, LifeScan Inc.).

Telemetry implanted mice tested for salt-sensitivity in experiment 1 were subsequently subjected to acute experiments to assess sensitization to restraint-stress (Fig 1). First, we evaluated the cardiovascular responses to an acute immobilization protocol. WT and Ren-b^Null mice fed high salt diet were placed for 10 minutes in a restrainer, while telemetric blood pressure and heart rates were monitored. Then, the diet was switched back to a low salt diet and the same procedure was performed 14 days later. Baseline telemetric blood pressure and heart rate were acquired for 10 minutes before mice were placed in the restrainer. Once in the restrainer, data were acquired for 10 additional minutes. A separate cohort of males and females fed with a normal salt diet was used to study the metabolic responses to restraint-stress. Blood glucose was measured at baseline before restraint-stress and induced a restrain-stress for 30 minutes. Finally, mice were released to the home cage and blood glucose was measured again during recovery. Every 15 minutes blood samples were obtained from tail veins in conscious mice and blood glucose levels were determined using a glucometer (One Touch Ultra 1, LifeScan, Inc.). Mice were sacrificed by decapitation at 30 minutes post-recovery phase and blood was collected for renin and glucocorticoid determinations.