4.1 Animals and experimental design
Thirty-four male C57BL/6 mice (5-6 weeks old) were purchased from Department of Laboratory Animal Science Fudan University (Shanghai, China). Mice were housed under controlled temperature (22 ± 2°C) and humidity (55 ± 5%) and under a light/dark cycle of 12:12 hours with free access to water and food. After one week of adaptive feeding, the mice were randomly divided into two groups: Normal chow diet (n=10), High-fat diet (n=24). After eight weeks, the mice were intraperitoneally injected with streptozotocin (40mg/kg/d, for five consecutive days, dissolved in citrate buffer) for the High-fat diet group. For Normal chow diet group, the mice were intraperitoneally injected with the same amount of citrate buffer (40mg/kg/d, for five consecutive days). Blood glucose of mice was measured weekly, and mice (n=24) with random blood glucose ≥16.7mmol/L in HFD group were included in the next step of experiment.
Mice in HFD group were randomly divided into two groups: T2DM group (n=12) and T2DM+Empagliflozin group (n=12), while Normal chow diet group was renamed as Control group (n=10). For T2DM+Empagliflozin group, mice were administered Empagliflozin (10mg/kg/d, dissolved in normal saline) by gavage daily for 6 weeks. For Control and T2DM group, mice were administered the same amount of normal saline (10mg/kg/d) by gavage daily for 6 weeks. After above treatment, the mice were sacrificed and the tissue samples and stool samples were collected. All procedures are carried out in accordance with health guidelines of the National Institutes of China on animal care and use, and were approved by the Animal Ethics Committee of Fudan University.
Oral glucose tolerance test (OGTT) and intraperitoneal glucose tolerance test (IPGTT)
OGTT and IPITT were conducted as the previous study described18. For OGTT, mice were fasted for 12 hours and orally gavaged 2 g/kg glucose. The blood sample was collected from the caudal vein at intervals of 0, 15, 30, 60, 120 minutes after glucose administration. Blood glucose level was measured immediately by a blood glucose meter (Elite, Bayer). On another day, IPITT was performed after fasting for 12 hours. The mice were injected intraperitoneally with 0.75 U/kg of insulin, and the caudal vein blood glucose levels were measured at 0, 15, 30, 60, 120 minutes after insulin injection. The area under the curve (AUC) of OGTT and IPITT were calculated based on the measured data.