2DG Uptake Assay Kit

Background

Measuring uptake of 2-deoxyglucose (2DG) in tissues and cells is a reliable approach method to estimate the amount of glucose uptake and thereby to explore the regulation of glucose metabolism and mechanism of insulin resistance. Radioisotope-labeled 2DG is usually used to measure 2DG uptake both in vivo and in vitro. However a specialized RI facility with strict limitations is required to carry out the radioisotope (RI) method and cannot be handled in ordinal laboratories. Furthermore, radioactive 2DG administered into cultured cells remains in extracellular space, and therefore the results obtained must be corrected by separating the extracellular 2DG and intracellular 2DG/2DG-6-phosphate (2DG6P) in the cells.
This kit is based on the enzymatic method for the direct measurement of amount of 2DG6P  without the use of radioisotope materials (Saito K and Minokoshi Y, et al. Analytical Biochem 412: 9-17, 2011).

A small amount of 2DG is administered into animals or cultured cells, and endogenous glucose and glucose-6phosphate (G6P) in tissues or cells is oxidized in the presence of a low concentration of G6PDH. 2DG-6-phosphate (2DG6P) accumulated in cells is then oxidized in the presence of a high concentration of G6PDH. NADPH produced from 2DG6P and G6PDH is quantified at 420 nm with the use of a recycling amplification enzymatic-photometric system. The novel enzymatic method can quantify 2DG or 2DG6P in the range of 5-80 pmol. As all enzyme reactions are performed in one 96-well microplate by the sequential addition of reagents, this method can be adopted for industrial robots. This method is useful for the screening of anti-diabetic drugs as well as for research in glucose metabolism and insulin signaling.

2-Deoxyglucose (2DG) Uptake Measurement Kit flyer download [PDF]

Principle

A recycling enzymatic amplification system measures NADPH produced by the in vitro oxidation of 2DG6P accumulated in cells following 2DG uptake.

1) So as not to effect glucose metabolism, only a small amount of 2DG is added to live cells. Incorporated 2DG is converted by cell metabolism to 2D6GP, which accumulates in cells. Cell lysates are then prepared.

2) To eliminate detection of G6P, G6P is oxidized (to 6PG) with NAD+ and a low concentration of G6PDH.

3) 2DG6P levels are quantitated by measuring the amount of NADPH produced during 2DG6P oxidation
(with NADP+ and a high concentration of G6PDH) in a phtometric recycling amplification/detection system

All reaction steps are conveniently performed in a single well by the sequential addition of premixed reagents. Ideal for assay automation

Features and Advantages

☆ No RI materials are required, and 2DG uptake can be measured in any ordinal laboratories.
☆ Direct measurement of 2DG6P amount accumulated in target cells.
☆ High sensitivity with the use of enzyme-recycling amplification reaction.

< Comparison between this kit and RI method >

Application

2-Deoxyglucose (2DG) Uptake Measurement kit Manual PDF

< Temporal change of O.D. for different concentrations of 2DG6P >

< Experimental example 1 – 2-deoxyglucose (2DG) uptake by 3T3-L1 cells>

< Experimental Example 2 - 2-deoxyglucose (2DG) uptake by human adipocytes >

   

<Reference>
1. Kumiko Saito, Suni Lee, Tetsuya Shiuchi, Chitoku Toda, Masahiro Kamijo, Kyoko Inagaki-Ohara, Shiki Okamoto, Yasuhiko Minokoshi (2011).  An enzymatic photometric assay for 2-deoxyglucose uptake in insulin- responsive tissues and 3T3-L1 adipocytes. Anal.Biochem., 412, 9-17.
2. Bo M. Jørgensen and Hans N. Rasmussen (1979). Recycling analysis of nicotinamide-adenine dinucleotide phosphates (NADP and NADPH). Anal.Biochem., 99, 297-303