Insuman rapid

HepG2 (obtained from ATCC, ATCC-HB-8065) and LX-2 (Sigma-Aldrich, St. Louis, United States, SCC064) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-ThermoFisher Scientific Waltham, United States), supplemented with 10% FBS, 2 mM L-glutamine, 100 units/ml penicillin, and 0.1 mg/ml streptomycin, and incubated in a 5% CO₂ humidified incubator at 37 °C. HepG2 and LX-2 were exposed to 0.33 µM insulin (Insuman Rapid, Sanofi Aventis) for 6 h.
In another experimental setting, hsa-miR-101-3p mimic (# 4464066) or miR-1 positive control (# 4464062) (mirVana miRNA mimic, ThermoFisher Scientific) were administered at a final concentration of 25 nM in complete medium without antibiotics and 0.5% bovine serum albumin (BSA). The cells were washed with PBS and then transiently transfected with 25 nM mir-101 mimic or miR-1 positive control using Lipofectamine 3000 (Thermo-fisher Scientific) according to the manufacturer’s instructions. We tested two different miRNA mimic concentrations (25 and 50 nM) and reached a great efficiency of transfection with both concentrations. Thus, we chose the lower concentration (25 nM), which has been frequently used in various studies [17 (link),18 (link),19 (link)]. At least three independent experiments were performed.

Prior to both tests, mice were fasted for 5 h and further deprived of food and water for the duration of the experiments. The animals were restrained, and tail veins were punctured. Blood glucose was measured with a glucose meter (Aviva Accu Check, Roche, Mannheim, Germany), representing baseline levels. For the insulin tolerance test, the insulin stock solution (40 U/mL; Insuman Rapid, Sanofi-Aventis, Frankfurt, Germany) was diluted to 1 U/mL in sterile saline solution. Mice were injected intraperitoneal with 1 U/kg body weight. For the glucose tolerance test, mice were injected with 2 g/kg body mass of D-glucose (20% w/v in sterile saline solution). For both experiments blood glucose was measured after 5, 10, 15, 20, 25, 30, 45, 60, 80, 100 and 120 min after injection.

Plasma triglyceride and total cholesterol were measured using the ILAB 600 Analyzer (Instrumentation Laboratory, Bedford, MA, USA). Fasting blood glucose concentrations were determined by a glucometer (GlucoMen LX meter, Menarini, Florence, Italy). Intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) were carried out in overnight-fasting mice. For IPGTT, mice were injected intraperitoneally (i.p.) with glucose (2 g/kg b.w.) (D-glucose, Sigma-Aldrich, Milan, Italy) in 0.9% saline. For ITT, mice were injected i.p. with insulin (0.5 U/kg b.w.) (Insuman Rapid, Sanofi Aventis, Italy) in 0.9% saline. Tail-vein-measured glucose concentrations were taken at different time points (0, 15, 30, 60, and 120 min). Plasma insulin was quantified using a mouse ELISA kit (Alpco diagnostics, Salem, NH, USA) according to the manufacturer’s instructions, and the HOMA-IR, index of insulin resistance, was calculated as the ratio of fasting insulin (ng/mL) and fasting glucose (mg/dL) divided by the constant 22.5.