Freestyle glucometer

A model of hyperglycemia was generated by feeding 5 week old female C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) a 60% kCal fat diet (Harlan Teklad, Indianapolis, IN) for 4 months. Normo- and hyperglycemic mice were then treated with metformin (200 mg/kg/day) or placebo (PBS) intraperitoneally for 3 weeks. ID8 mouse ovarian cancer cells (1.2×10⁶) were injected orthotopically into the ovarian bursa and treatment with metformin or placebo was continued for 12 weeks before the mice were sacrificed. Glucose tolerance tests were performed following a 16 h fast by injecting 2 g/kg D-glucose (Sigma-Aldrich) intraperitoneally and measuring blood glucose levels with a FreeStyle glucometer (Abbott Laboratories). Mean tumor weight in the ovary was compared between normo- and hyperglycemic mice treated with metformin or placebo. Tumors were homogenized in RIPA buffer and protein expression was analyzed by western blot. All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Chicago.

Mice were fasted for 3–4 h prior to each experiment. They were induced with 2.5% isoflurane and maintained at 1.7%. Mice were placed in the supine position, and a midline cervical incision was made. The left cervical vagus nerve was isolated from the carotid bundle. The extraneural sheath overlaying the nerve was removed to ensure good contact with the leads, and the nerve was placed in a cuff electrode with a 200 μm inner diameter (PDMS Sling μCuff, CorTec). The neural recording was acquired using Plexon Omniplex Neural Data Acquisition system, with a sampling rate of 40 kHz. A ground electrode was placed next to the right salivary gland. The nerve was kept from desiccation by placing parafilm over the recording area. Following acquisition of baseline activity for 30 min, animals were treated with insulin (6 mg/kg Humulin-R U-100, Eli Lilly Medical) or glucose (2 g/kg) intraperitoneally (i.p.), and recordings were continued for 30 min post-injection. Glucose measurements were taken before and throughout the recording via tail nick and Abbott Freestyle glucometer.

Intraperitoneal glucose tolerance tests (ipGTT) and insulin tolerance tests (ipITT) were performed on weeks 12 and 13, respectively. Mice were food-deprived for six hours before each test. The IpGTT was initiated by an intraperitoneal injection of 2 g/kg of glucose and the ipITT by an intraperitoneal injection of 0.5 UI/kg of insulin. Glucose was measured in blood from the tail vein at 0, 15, 30, 45, 60, 90, and 120 min after the injection using a portable FreeStyle glucometer (Abbott, Mexico City, Mexico).

Blood was collected between 8AM and 10AM in both fed and 16h-fasted state. Glycaemia was determined from tail vein blood from awake animals using the Freestyle glucometer (Abbott, Switzerland). Plasma insulin was measured from blood samples collected by cardiac puncture (400ul) into heparinized tubes from euthanized 12 and 16 week-old mice. Insulin levels were determined by using an insulin enzyme immunoassay kit (Mercodia).