Diabetic Mouse Model Development

Male ApoE^−/− mice were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd (China) and housed in an environmentally controlled room at 22 ± 2°C and 50% ± 5% humidity with a 12 h/12 h light/dark cycle with free access to food and water. They were allowed to acclimate one week before performing intraperitoneal glucose tolerance test (IPGTT) and then were randomly assigned to control group (Chow group) and diabetic group (DM group). Mice in the diabetic group were fed with a high fat diet (20% fat, 20% sugar, and 1.25% cholesterol; Beijing HFK Bioscience company, China). After 8 weeks, IPGTT was performed to confirm the appearance of insulin resistance. Those mice showing insulin resistance were intraperitoneally injected with one low-dose STZ (75-80 mg/kg body weight in 0.1 mol/L citrate buffer, pH 4.5). Two weeks after STZ injection, most mice displayed hyperglycemia, insulin resistance, and glucose intolerance (Supplementary Figure S1), as previously reported (Wang et al., 2014 (link)). Mice with similar degrees of hyperglycemia and body weight were randomly divided into DM (DM, n = 20), DM + nc-shRNA (n = 20) and DM + ALK7-shRNA, n = 20), and injected with nc-shRNA or ALK7-shRNA lentivirus at a dose of 4×10⁹ ifu per mouse via tail vein. At the end of experiment, mice were sacrificed and aortas were collected for subsequent analysis.