An enzymatic photometric assay for 2-deoxyglucose uptake in insulin-responsive tissues and 3T3-L1 adipocytes
An enzymatic photometric assay for 2-deoxyglucose uptake in insulin-responsive tissues and 3T3-L1 adipocytes
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DOI：
10.1016/j.ab.2011.01.022
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发表时间：
2011-05-01
影响因子：
2.9
通讯作者：
Minokoshi, Yasuhiko
中科院分区：
文献类型：
--
作者：
Saito, Kumiko;Lee, Suni;Minokoshi, Yasuhiko
采用96孔微孔板进行光度分析的酶法测定胰岛素敏感组织和分化的313-L1脂肪细胞的2-脱氧葡萄糖（2DG）摄取。对于体内测量，将少量非放射性标记的2DG注射到小鼠中而不影响葡萄糖代谢。为了对细胞中积累的少量2-脱氧葡萄糖6-磷酸（2DG 6P）进行光度定量，我们将葡萄糖-6-磷酸脱氢酶、谷胱甘肽还原酶和5，5 '-二硫代双（2-硝基苯甲酸）引入NADPH的再循环扩增反应中。我们优化的酶反应完全氧化内源性葡萄糖6-磷酸（G6 P）和葡萄糖在小鼠体内组织和血清以及在313-L1脂肪细胞在体外。通过连续加入试剂在一个96孔微孔板中进行所有反应，并且该测定能够定量5-80 pmol范围内的2DG和2DG 6P。在体外和体内的胰岛素刺激的存在或不存在的情况下，2DG摄取的测定所获得的结果与标准放射性同位素方法所获得的结果相似。因此，酶法测定应被证明是有用的，在体内以及在培养的细胞中的胰岛素反应组织中的2DG摄取的测量。(C)2011 Elsevier Inc. All rights reserved.
An enzymatic assay adapted to photometric analysis with 96-well microplates was evaluated for the measurement of 2-deoxyglucose (2DG) uptake in insulin-responsive tissues and differentiated 313-L1 adipocytes. For in vivo measurements, a small amount of nonradiolabeled 2DG was injected into mice without affecting glucose metabolism. For photometric quantification of the small amount of 2-deoxyglucose 6-phosphate (2DG6P) that accumulates in cells, we introduced glucose-6-phosphate dehydrogenase, glutathione reductase, and 5,5'-dithiobis(2-nitrobenzoic acid) to the recycling amplification reaction of NADPH. We optimized the enzyme reaction for complete oxidation of endogenous glucose 6-phosphate (G6P) and glucose in mouse tissues in vivo and serum as well as in 313-L1 adipocytes in vitro. All reactions are performed in one 96-well microplate by consecutive addition of reagents, and the assay is able to quantify 2DG and 2DG6P in the range of 5-80 pmol. The results obtained with the assay for 2DG uptake in vitro and in vivo in the absence or presence of insulin stimulation was similar to those obtained with the standard radioisotopic method. Thus, the enzymatic assay should prove to be useful for measurement of 2DG uptake in insulin-responsive tissues in vivo as well as in cultured cells. (C) 2011 Elsevier Inc. All rights reserved.