Freestyle freedom lite

Mice were fasted for 12 to 14 h by removing the feeding tray and housing in fresh bedding with fasting trays. All mice had ad libitum access to plain drinking water or drinking water containing erythromycin ethylsuccinate (equivalent to 20 mg/kg) during the fasting period. Glucose (2 g/kg of body mass, dissolved in sterile 0.9% sodium chloride) was administered by intraperitoneal injection and blood glucose was measured from the tail tip using a glucometer (Abbott Freestyle Freedom Lite, Australia). Blood glucose measurements were performed at 0 (basal level), 15, 30, 60, and 120 min after glucose administration.

Venous blood was drawn from the tail vein. Blood glucose levels were measured using the glucose dehydrogenase flavin adenine dinucleotide method (FreeStyle Freedom Lite glucose monitor; Abbott Japan, Chiba, Japan).

Male Wistar rats were anesthetized with urethane (40 μg·kg⁻¹, i.p.). Following tracheal cannulation, the anesthetized rats were mechanically ventilated, and the femoral artery was cannulated for continuous monitoring of mean arterial blood pressure and heart rate. Mean pulmonary artery pressure (mPAP) was measured in the PA of open chest rats. Hemodynamic parameters were recorded with a pressure transducer (ADInstruments Powerlab/4SP, ML 750, USA) connected to a pressure processor amplifier (Animal Bio Amp, ADInstruments, FE 136) and signal conditioner (ADInstruments, FE 221). Non fasting blood glucose measurements were obtained through a blood drop from a cannulated femoral artery using a handheld glucometer and One-Touch glucometer strips (FreeStyle Lite and FreeStyle Freedom Lite, Abbott, USA). After the blood had been collected from the femoral arteries into heparinized tubes, all experimental rats underwent saline perfusion followed by removal of the right middle lung which was stored at −80 °C until Western blot analysis. Perfusion with 10% formalin was then performed followed by removal of the right lower lung and heart which were soaked in 10% formalin and stored at 4 °C for future tissue sampling and assessment of the degree of right ventricular hypertrophy.

Fasting blood glucose levels were measured by a glucometer (FreeStyle Freedom Lite, Abbott, Oslo, Norway) from the ventral tail artery from female mice after 3 h of fasting. For insulin and C-peptide measurements of female mice, the total blood volume was collected from the chest cavity immediately after cardiac excision. The blood was left at room temperature for 15–20 min to coagulate, before centrifugation at 1500 rcf at 4°C for 10 min. The blood serum was collected and immediately stored at −20°C. Insulin and C-peptide serum concentrations were determined by a mouse insulin ELISA (80-INSMS-E01, Alpco, NH, United States) and mouse C-peptide ELISA (80-CPTMS-E01, Alpco).

Mice were fasted for 4 h during the light phase. Two types of GTTs were performed. Oral GTT (oGTT) where mice were orally gavaged with 20% glucose (Braun, Germany) (2 g/kg) and an intraperitoneal GTT (i.p. GTT) where mice were intraperitoneally injected with 20% glucose solution (2 g/kg). Blood glucose levels were measured from tail tip punctures using a Freestyle Freedom Lite glucometer (Abbott, Chicago, Illinois, USA) at the 0, 15, 30, 60, 90 and 120 min time points.