Novolin

TG clearance was assessed in mice fasted overnight (1700–0900 h) after an oral gavage of 200 μL of food-grade olive oil (Oleoestepa, Estepa, Spain). The rate of hepatic VLDL-TG secretion was assessed after tyloxapol injection (500 mg/kg i.p.; Sigma-Aldrich). Glucose tolerance tests were performed in mice fasted overnight that were injected with 2 mg glucose/g BW i.p. Insulin tolerance tests were performed in mice 4 h after food removal, starting at 0700 h by injecting 1.5 mU insulin/g BW i.p. (Novolin; Novo Nordisk, Bagsvaerd, Denmark). Blood samples were taken from lateral tail vein, and blood glucose measured (AlphaTRAK 2; Abbott Laboratories, Abbott Park, IL). To assess ex vivo WAT lipolysis, 50–60 mg of tissue from urogenital fat pads was washed in cold PBS and minced into small pieces, then incubated in 500 μL Krebs-Ringer HEPES buffer without or with 1 µmol/L isoproterenol for 2 h at 37°C 5% CO₂. Media were collected to measure glycerol production (Free Glycerol Reagent; Sigma-Aldrich). Hepatic insulin sensitivity was assessed in mice fasted overnight that were injected with saline or 2 mU/g BW insulin i.p. Twenty minutes later, the liver was collected for Western blot analysis.

Blood glucose concentrations were determined using an AlphaTrak glucose meter (Abbott Laboratories). Glucose tolerance tests were performed on animals fasted overnight by intraperitoneally injecting glucose (2 mg/kg), as previously described (30 (link),31 (link)). Plasma insulin concentrations were determined using a Mouse Insulin ELISA kit (ALPCO). For an insulin tolerance test, animals fasted for 6 h received an intraperitoneal injection of either saline or human insulin (0.5 units/kg; Novolin; Novo Nordisk). Fasting glucose and insulin were measured after an overnight fast.