Accu check advantage

Mbd2 knockout (Mbd2^−/−) mice in the C57BL/6 background were kindly provided by Dr. Adrian Bird (Edinburgh University, UK) [41 (link)]. The Mbd2 deficient NOD (Mbd2^−/− NOD) mice were generated by backcrossing the Mbd2^−/− C57BL/6 with NOD/ShiLtJ mice (purchased from Jackson Laboratory) for more than 20 generations, and their purity of NOD background was confirmed by DNA sequencing. NOD.scid mice were purchased from Beijing HFK Bioscience (Beijing, China). All mice were housed in a specific pathogen-free animal facility at the Tongji Medical College on a 12/12 h light/dark cycle. After 5 weeks of age, the NOD mice were monitored for blood glucose two times per week using an Accu-Check Advantage glucometer (Roche Diagnostics, Indianapolis, IN, USA), and classed as diabetic once two consecutive blood glucose readings were >13.8 mmol/l [42 (link)]. All protocols for animal studies were approved by the Tongji Hospital Animal Care and Use Committee (TJH-201612001) in accordance with the National Institutes of Health guidelines.

Glucose tolerance tests (GTTs) and insulin tolerance tests (ITTs) were carried out as described elsewhere (11 (link)). In brief, 6 h after fasting, mice were intraperitoneally (i.p.) injected with 1 g/kg body weight of glucose solution. At 0, 30, 60, and 120 min blood glucose levels were measured by a glucometer (AccuCheck Advantage; Roche Diagnostics GmbH, Mannheim, Germany).
Four hours after fasting, ITT was performed. Briefly, human insulin (Sanofi-Aventis, Frankfurt, Germany) 1 U of insulin/kg body weight was i.p. injected and at 0, 30, 60, and 120 min blood glucose levels were measured. The area under the curve (AUC) was derived by calculating the area between the x-axis and a given curve using GraphPad Prism software (version 8.3; GraphPad Software, San Diego, Calif., USA).
Lipid profiles and liver enzymes—serum glutamic-oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT)—were measured using Reflotron (Roche Diagnostics GmbH) according to the manufacturer's protocol.

For intraperitoneal insulin tolerance testing (IPITT), mice were fasted for 3–4 h before the administration of insulin. Insulin was prepared at 100 iu/ml in normal saline and used to resuspend nitrogen-dried 2-[1,2-³H(N)]-deoxy-D-glucose (0.37 MBq). Basal blood glucose was measured in tail blood using an Accu-Check Advantage meter (Roche Diagnostics, Burgess Hill, West Sussex, UK). A further 10 µl of blood was collected in a microfuge tube containing 1 iu heparin in saline, mixed and placed on ice. Immediately afterwards, insulin and deoxyglucose tracer was administered intraperitoneally at a dose of 0.75 iu/kg. At 15, 30, 60 and 90 min after insulin administration, blood glucose was measured, as described above. After taking the final blood sample, the mice were euthanised by cervical dislocation and tissues were dissected, weighed and frozen in liquid nitrogen-cooled isopentane. Glucose clearance into TA muscles was determined as described previously^{42 (link)}.

To induce diabetes, a single high dose of streptozotocin (STZ; 225 mg/kg; Sigma-Aldrich) was intraperitoneally injected into C57BL/6 mice (fasted for 16 h beforehand, body weight 20–23 g). Every week after STZ administration, serum glucose levels were measured using an Accu-Check Advantage glucometer (Roche, Indianapolis, IN, USA) during nonfasting status. Mice with a plasma glucose level >200 mg/dl at 3 weeks after injection were regarded as having STZ-induced diabetes [16 (link)].

Hydration status was assessed by urine specific gravity (USG) (503 Nippon Optical Works, Japan) before and at the completion of exercise. Blood samples were taken from the middle digit of the hand at rest, 5 km intervals throughout each time trial and immediately post exercise for the determination of blood lactate concentration [La⁻] (Lactate Pro, ARKRAY, Kyoto, Japan) and glucose concentrations [Glu] (Accu-check Advantage, Roche Diagnostics Corporation, Indianapolis, USA). Heart rate was continuously monitored (Polar Electro OY M61, Kempele, Finland) and recorded at 2 km intervals. The RPE [2] (link), [17] (link) was recorded every 5 km, before and after each sprint and when EMG was recorded.
Core temperature (T_c) was measured using the CoreTemp Telemetry System (HQInc. Wireless Sensing System and Design, Palmetto, Florida, USA). Participants ingested the telemetry pill the night before the trial and were advised by the researcher at what time it should be ingested according to the participants bowel motion. Skin temperature was monitored with skin thermistors attached at four sites (chest, arm, thigh, leg) and connected to a digital telethermometer (Zencor/Zentemp 5000, Victoria, Australia) and recorded every 2 km. Mean skin temperature (T_sk) was calculated as previously described [3] (link), [18] (link).