Animal experiments
All animal use was approved by the Animal Care and Ethical Committee of
Nanjing Medical University. C57BL/6J mice [RRID: IMSR_JAX:000664]
(male, 8-week old, 22 ± 2 g) were obtained from Animal Resource Center
(Nanjing Medical University, Nanjing, China). Mice were housed under a
12 h light, 12 h dark cycle, with food and water freely accessible.
After a 4-hour fast, STZ (Sigma-Aldrich, St. Louis, MO, USA) diluted in
Na-citrate buffer (PH = 4.5) was injected intraperitoneally at a dose of
50 mg/kg for consecutive 5 days to destroy the islet beta cells of the
mice. Hyperglycemia was defined as 2 consecutive random blood glucose
readings of 16.7 mmol/L or higher.
For heparanase inhibition, OGT2115 (Tocris, Minneapolis, MN, USA) was
dissolved in dimethyl sulfoxide (DMSO) and diluted to different
concentrations with sterile water containing 5% Tween 80 and 30%
PEG400. STZ mice received vehicle (sterile water containing 1% DMSO,
5% Tween 80, and 30% PEG400), a low OGT2115 dosage (3 mg/kg) or a
higher dosage (10 mg/kg). OGT2115 and vehicle were subcutaneous injected
daily for 4 weeks. The food intake, body weight gain and random blood
glucose levels of the mice were followed up every week. All animal
experiments were performed and analyzed through blinded experimenters.
Each mouse in STZ or control group was numbered randomly within the
weight range. After being divided randomly in each group, the animals
were given their permanent number in the cages.
Intraperitoneal Glucose tolerance test (IPGTT) and
insulin measurement
After the 4-week drug administration period, the animals were fasted for
12 hours and glucose (2 g/kg of body weight dissolved in saline) was
intraperitoneally injected. Blood glucose was then measured from tail
vein blood with a glucometer (Roche Diagnostics, Indianapolis, IN, USA)
and test strips at 0, 30, 60, 90, and 120 min following the glucose
injection. The results were displayed as a blood glucose curve and the
area under the curve (AUC). Before and 30 min after glucose loading,
additional blood samples were collected and serum insulin concentrations
were measured using insulin ELISA kits (EZassay, Shenzhen, China).