Glucometer

Fasting blood glucose levels were measured by using a Glucometer (OMRON Corporation, Tokyo, Japanese). For urine collection, mice were held in a metabolic cage for 24 h. Levels of urinary albuminuria and podocalyxin were measured using ELISA kits (Elabscience, Wuhan, China). Creatinine was tested by a commercial assay kit (Jiancheng, Jiangsu, China).

Glucose tolerance test (GTT) was performed as previously described14 (link). Briefly, mice were fasted overnight and intraperitoneally administered glucose (2 g kg⁻¹). Blood glucose levels were measured by using a glucometer at the indicated time points (Omron, Beijing, China). Plasma TG analysis: fasting plasma TG was determined with reagent from Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacture's instruction.

The progeny mice at 4 and 10 weeks of age were fasted for 12 h with free access to water only, and their tail-vein blood was collected to measure fasting blood sugar with an Omron glucometer. All mice were intraperitoneally injected with glucose at a dose of 2 g/kg, and their blood glucose levels were also measured at 30, 60, 90, and 120 min after injection. Orbital blood samples were collected from each of the mice by removing the eyeball and serum was separated. Triglycerides (TG), total cholesterol (T-CHO), high-density lipoprotein (HDL), low-density lipoprotein (LDL), and insulin (INS) were measured using commercially available kits (Jiancheng Biotech, China) according to the manufacturer’s protocols.

The glucose tolerance test (GTT) was carried out after 4 weeks of treatment. Mice were oral administrated with 2 g/kg body weight of D-glucose (Sigma-Aldrich, St. Louis, MO, USA) after 16 h fast. Blood samples were taken from the tail vein of the mice at 0, 30, 60, 90 and 120 min after glucose administration, respectively. The GTT was carried out on awake mice without anesthetization. The glucose levels in serum were also determined by a glucometer (OMRON (China) Co., Ltd, Beijing, China).

Mouse metabolic assays were performed. Briefly, body weight, fasting blood glucose (FBG) levels and fasting serum insulin (FINS) levels were examined after a 6 h fasting treatment by using a glucometer (OMRON, Japan) and by ELISA (ExCell Bio, Shanghai, China), respectively. And the homeostatic model assessment indices of insulin resistance (HOMA-IR) was calculated with the equation (FBG (mmol L⁻¹) x FINS (mIU L⁻¹))/22.5. To perform the glucose tolerance tests, 1.5 g kg⁻¹ glucose (Sigma-Aldrich, St Louis, MO, USA) was i.p. injected into mice, whereas 0.75 U kg⁻¹ insulin (Novolin R, Novo Nordisk, Bagsvaerd, Denmark) was i.p. injected into mice for insulin tolerance tests. Blood glucose levels were examined at 0, 15, 30, 60, and 120 min after injection. The AUC are given as the incremental area under the curve, calculated by the conventional trapezoid rule.

After 12 h fasting treatment, mice fasting blood glucose (FBG) levels and fasting serum insulin (FINS) levels were examined via using a glucometer (OMRON, Japan) and by ELISA (ExCell Bio, Shanghai, China), respectively. And the homeostatic model assessment indices of insulin resistance (HOMA-IR) was calculated with the equation (FBG (mmol/l) × FINS (mIU/l))/22.5. To perform the glucose tolerance tests, 2 g/kg glucose (Sigma-Aldrich, St Louis, MO, USA) was intraperitoneal (i.p.) injected into mice, whereas 0.75 U/kg insulin (Novolin R, Novo Nordisk, Bagsvaerd, Denmark) was i.p. injected into mice for insulin tolerance tests. Blood glucose levels were examined at 0, 15, 30, 60, 90, and 120 min after injection and serum sample was collected from eye canthus blood at 0, 5, 15, and 30 min after glucose injection. Insulin level was evaluated using mice insulin ELISA kit (Crystal Chem, USA), according to the manufacturer’s instructions. The AUC, calculated by the conventional trapezoid rule, are given as the incremental area under the curve.

All animal protocols were approved by the University Committee on Use and Care of Animals of Huazhong University of Science and Technology. STZ is widely used to produce DM model in rats (Furman, 2021 (link)). As previously described with some modifications (Wojcicka et al., 2010 (link)), STZ (Sigma) was dissolved in 0.1 M of a citrate buffer (pH 4.5), and injected intraperitoneally into 5-week-old male Sprague-Dawley rats at a dose of 50 mg/kg body weight after overnight fasting. Rats in control group were injected with citrate buffer. Five days after STZ injection, fasting glucose (IFG) level in the blood obtained from the tail was measured using a glucometer (Omron, Kyoto, Japan). IFG >11.1 mmol/L and stable for 2 weeks were considered diabetes.