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An enzymatic photometric assay for 2-deoxyglucose uptake in insulin-responsive tissues and 3T3-L1 adipocytes

Authors :
Saito, Kumiko
Lee, Suni
Shiuchi, Tetsuya
Toda, Chitoku
Kamijo, Masahiro
Inagaki-Ohara, Kyoko
Okamoto, Shiki
Minokoshi, Yasuhiko
Source :
Analytical Biochemistry. May2011, Vol. 412 Issue 1, p9-17. 9p.
Publication Year :
2011

Abstract

Abstract: 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 3T3-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 3T3-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 5–80pmol. 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. [Copyright &y& Elsevier]

Details

Language :
English
ISSN :
00032697
Volume :
412
Issue :
1
Database :
Academic Search Index
Journal :
Analytical Biochemistry
Publication Type :
Academic Journal
Accession number :
59167238
Full Text :
https://doi.org/10.1016/j.ab.2011.01.022