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 hepa tic glucose production(HGP)using a fuorescent D-glucose analog,2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglu-cose(2-NBDG).To prove the reliability and feasibility of this method,whole-body insulin sensitivity was compared between conscious normal ICR mice and diabetic KK^(Ay) 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.

Association between four adipokines and insulin sensitivity in patients with obesity, type 1 or type 2 diabetes mellitus,and in the general Chinese population

Background Hyperinsulinemic euglycemic clamp is the gold standard to evaluate the insulin sensitity, but it is too complicated and expensive to use in clinic.We tried to find an alternative indicator to reflect insulin sensitivity.To evaluate the association between the four adipokines, adiponectin, leptin, resistin and tumor necrosis factor-α （TNF-α） with insulin sensitivity, we used a hyperinsulinemic euglycemic clamp to test insulin sensitivity in Chinese patients with obesity and type 1 or type 2 diabetes mellitus versus controls.Methods In this parallel control study, we tested insulin sensitivity using a hyperinsulinemic euglycemic clamp in different groups, then examined levels of adiponectin, leptin, resistin and TNF-α in serum, and the relationship between the different adipokines and glucose disposal rate （M value）, as well as insulin sensitivity index （M value/insulin, M/I）,which are the ＂gold standard＂ indices of insulin sensitivity.Results There were significant differences in mean leptin values in the four adipokines from the four different groups （P〈0.001; comparison of the variation between different groups was analyzed by variance analysis）.Compared to controls （using multiple comparison two-way Dunnett t test）, only the leptin level showed significant differences in the four adipokines from the four different groups at the same time （P 〈0.001）.The association analysis between the different adipokines and M or M/I values also showed that only leptin negatively correlated with M （r=-0.64, P 〈0.001） or M/I values （r=-0.56, P〈0.001）; there was no relationship between the other three adipokines and M or M/I values.Conclusion Only leptin was associated with M or M/I values.Therefore, leptin might be one of the predictive factors of the degree of insulin resistance and risk of the accompanying disease.