The application of 2-NBDG as a fluorescent tracer for assessing hepatic glucose production in mice during hyperinsulinemic euglycemic clamp

Methods of performing insulin clamps vary between laboratories. Here we present a protocol of insulin clamping in conscious mice, with the significant advantage of avoiding multiple surgical catheterizations and non-physiologic metabolism during the induction of anesthesia. Using this technique we also established a new method for measuring hepatic glucose production (HGP) using a fluorescent d-glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxyglucose (2-NBDG). To prove the reliability and feasibility of this method, whole-body insulin sensitivity was compared between conscious normal ICR mice and diabetic KKAy mice using the insulin clamp. Basal and clamp HGP was compared between normal C57 mice and diabetic db/db mice by using the modified clamp with 2-NBDG as a tracer. The glucose infusion rate (GIR), an index of insulin sensitivity, was significantly lower in KKAy mice than normal ICR mice (6.2±1.3 mg/kg/min vs. 31.3±2.9 mg/kg/min, P<0.001). The db/db mice also showed higher basal hepatic glucose production (25.8±2.2 mg/kg/min vs. 16.7±2.5 mg/kg/min, P<0.05), higher clamp HGP after insulin suppression (7.3±1.0 mg/kg/min vs. 0 mg/kg/min, P<0.001), and lower GIR (71.6±2.8 mg/kg/min vs. 15.2±1.6 mg/kg/min, P<0.001) than that obtained with normal C57 mice. In conclusion, this is the first report of the application of 2-NBDG, rather than isotopic tracers, for the determination of HGP in vivo.

We present a protocol of insulin clamping in conscious mice, with the significant advantage of avoiding multiple surgical catheterizations and non-physiologic metabolism during the induction of anesthesia.Figure optionsDownload as PowerPoint slide