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-phy...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.展开更多
The sodium-dependent glucose transporters 2(SGLT2)plays important role in renal reabsorption of urinal glucose back to plasma for maintaining glucose homeostasis.The approval of SGLT2 inhibitors for treatment of type ...The sodium-dependent glucose transporters 2(SGLT2)plays important role in renal reabsorption of urinal glucose back to plasma for maintaining glucose homeostasis.The approval of SGLT2 inhibitors for treatment of type 2 diabetes highlights the SGLT2 as a feasible and promising drug target in recent years.Current methods for screening SGLT2 inhibitors are complex,expensive and labor intensive.Particularly,these methods cannot directly measure nonradioactive glucose uptake in endogenous SGLT2-expressing kidney cells.In present work,human kidney cells,HK-2,was incubated with a fluorescent D-glucose derivant 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose(2-NBDG)and the fluorescent intensity of 2-NBDG was employed to measure the amount of glucose uptake into the cells.By optimizing the passages of HK-2 cells,2-NBDG concentration and incubation time,and by measuring glucose uptake treated by Dapagliflozin,a clinical drug of SGLT2 inhibitors,we successfully developed a new assay for measuring glucose uptake through SGLT2.The nonradioactive microplate and microscope-based high-throughput screening assay for measuring glucose can be a new method for screening of SGLT2 inhibitors and implied for other cell assays for glucose measurement extensively.展开更多
基金This work was supported by the grants from the Key Project of the National Twelfth-Five Year Research Program of China and National S&T Major Special Project on Major New Drug Innovation(No.2012ZX09301002-004).
文摘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.
基金supported by Yunnan Provincial Science and Technology Department of China to WX(2017FA044 and 2013HA023)Ministry of Science and Technology of the People’s Republic of China-The National Key Research and Development Program(2017YFC1700906).
文摘The sodium-dependent glucose transporters 2(SGLT2)plays important role in renal reabsorption of urinal glucose back to plasma for maintaining glucose homeostasis.The approval of SGLT2 inhibitors for treatment of type 2 diabetes highlights the SGLT2 as a feasible and promising drug target in recent years.Current methods for screening SGLT2 inhibitors are complex,expensive and labor intensive.Particularly,these methods cannot directly measure nonradioactive glucose uptake in endogenous SGLT2-expressing kidney cells.In present work,human kidney cells,HK-2,was incubated with a fluorescent D-glucose derivant 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose(2-NBDG)and the fluorescent intensity of 2-NBDG was employed to measure the amount of glucose uptake into the cells.By optimizing the passages of HK-2 cells,2-NBDG concentration and incubation time,and by measuring glucose uptake treated by Dapagliflozin,a clinical drug of SGLT2 inhibitors,we successfully developed a new assay for measuring glucose uptake through SGLT2.The nonradioactive microplate and microscope-based high-throughput screening assay for measuring glucose can be a new method for screening of SGLT2 inhibitors and implied for other cell assays for glucose measurement extensively.
