I0908

HUVECs were kindly provided by Professor Bai and cultured in Dulbecco's modified Eagle's medium (DMEM, HyClone) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. HUVECs from passages 3 to 8 were used in this study. The appropriate glucose concentration and treatment time were determined by treating HUVECs with D-glucose (G7021, Sigma-Aldrich) at the following concentrations: 25 mM D-glucose (normal glucose group (NG)), 40 mM D-glucose (H1 group), 60 mM D-glucose (H2 group), and 80 mM D-glucose (H3 group) for 24, 48, 72, and 96 h. In addition, HUVECs were treated with 25 mM D-glucose+60 mM mannitol to exclude the effect of the osmotic pressure of 80 mM D-glucose. In the following experiments, HUVECs were treated for 96 h with D-glucose and/or insulin (I0908, Sigma-Aldrich) at the following concentrations: 25 mM D-glucose (normal glucose group (NG)), 60 mM D-glucose (high-glucose group (HG)), and 60 mM D-glucose+1.5 μg/mL insulin (HG+INS group). HUVECs were transfected with IRSp53-siRNA or IRSp53-overexpressing lentivirus according to the manufacturers' recommended procedures. Descriptions of the IRSp53-siRNA- and IRSp53-overexpressing lentiviruses appear in the Supplementary Information (available here).

For glucose tolerance test (GTT), 16-week-old mice were fasted overnight for 16 h and provided with water ad libitum. The next day, mice were housed in individual cages and allowed to acclimate for 2 h followed by i.p. injection of 1.0 g/kg glucose (G8270, Millipore). For insulin tolerance test (ITT), 16-week-old mice were fasted overnight for 16 hours in individual cages with free access to water. Insulin (0.8 U/kg, I0908, Sigma-Aldrich) was administered by i.p. injection. Blood samples were obtained from a tail nick, and blood glucose was measured at 0, 15, 30, 60, 90, and 120 minutes using a commercial glucometer (Accu-Chek® Guide, Roche).

Insulin tolerance tests (ITT) and glucose tolerance tests (GTT) were performed after 9 months of dietary intervention, when mice were 13 months of age, to examine systemic glucose homeostasis. We also performed ITT and GTT on the 4-month-old young reference group after 2 weeks of AIN-93M diet feeding, which allowed us to assess the effect of age on glucose homeostasis.
Prior to ITT and GTT mice were food-deprived for 4 or 12 h, respectively. Insulin (0.75 mU /g body weight, I0908, Sigma-Aldrich, St. Louis, MO, USA) or glucose (0.4 mg dextrose/g body weight, D9434, Sigma-Aldrich, St. Louis, MO, USA) solutions were administrated through intraperitoneal injection. Tail vein blood glucose levels were determined by glucometer (Breeze2, Bayer, Whippany, NJ, USA) at 0, 30, 60, 90, and 120 min after insulin or glucose administration. Due to the limited blood volume of mice, we did not collect blood samples to measure circulating insulin levels during the tests.