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.