A Refinement of the Akabayashi-Saito-Kato Modification of the Enzymatic Methods for 2-Deoxyglucose and 2-Deoxyglucose 6-Phosphate

Chi, Maggie; Manchester, Jill K.; Carter, J.G.; Pusateri, Mary Ellen; McDougal, David B.; Lowry, Oliver H.

doi:10.1006/abio.1993.1130

Cited by 10 publications

(3 citation statements)

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“…2-Deoxyglucose 6-phosphate (dGlc6P) was measured by using a high concentration of glucose 6-phosphate (Glc6P) dehydrogenase (from Leuconostoc mesenteroides) after removing Glc6P (29). ATP was measured by using hexokinase and Glc6P dehydrogenase as described (26).…”

Section: Methodsmentioning

confidence: 99%

Ras signaling in the activation of glucose transport by insulin.

Manchester

Kong

Lowry

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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An approach involving micro'ijection and microanalysis has been developed to investigate signaltransduction pathways involved in the hormonal control of metabolism. We have applied this strategy to investigate the role ofRas signaling in the acute activation of glucose transport by insulin in cardiac myocytes. Glucose transport activity was assessed by measuring the initial rate of accumulation of 2-deoxyglucose 6-phosphate (dGlc6P) in individual cells after incubation in 2-deoxyglucose. Insulin increased accumulation of dGlc6P by 3-to 4-fold, consistent with its stimulatory effect on glucose transport. Accumulation of dGlc6P was increased severalfold by microin'ecting the nonhydrolyzable GTP analogue, guanosine 5'-[ythioltriphosphate, which activates members of the Ras superfamily of GTP-binding proteins. Injecting activated Ha-Ras protein also mimicked insulin by increasing dGlc6P; whereas, iu'ecting a Ras protein lacking the COOH-terminal site of fatty acylation required for Ras function was without effect. Introducing the neutralizing Ras antibody Y13-259 into cells attenuated the effect of insulin. These findings implicate Ras in the acute regulation of metabolism by insulin.Blood glucose levels are maintained within a relatively narrow range through the actions of several hormones, of which insulin is the most important (1). A rise in blood sugar promotes release of insulin, which stimulates glucose transport in skeletal muscle fibers, cardiac myocytes, and adipocytes (2, 3). Insulin increases glucose transport by causing translocation of glucose transporter isoform 4 (GLUT4) from intracellular compartments to the plasma membrane (4, 5). The effect on GLUT4 translocation reaches a maximum within minutes of an increase in the insulin concentration. Several other processes are rapidly changed in response to the hormone (6, 7). For example, glycogen synthesis is increased in skeletal muscle and fat cells, lipogenesis is increased in adipocytes and hepatocytes, and gluconeogenesis is decreased in hepatocytes. These acute metabolic actions of insulin are essential for the efficient uptake and storage of excess blood glucose.Insulin elicits other actions that develop with time courses of many minutes to hours (6). Such effects are observed in a wide variety of cell types and are associated with increased growth and/or differentiation of cells. There is increased evidence that these actions of insulin involve Ras, a GTP-binding protein that is a transducer of diverse physiological signals (8, 9). Insulin increases the proportion of Ras in the active GTP-bound state (10-12) via a mechanism that appears to involve formation of a complex between insulin receptor substrate 1 (IRS-1), guanine nucleotide-releasing factor mSOS, and growth factor receptor-bound protein (GRB2) (13)(14)(15)(16) (22). However, the potential of microinjection in investigating the important metabolic actions of insulin has not been exploited because of problems associated with measuring responses in the cells that have been injected. ...

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Section: Methodsmentioning

confidence: 99%

Ras signaling in the activation of glucose transport by insulin.

Manchester

Kong

Lowry

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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“…To quantify 2DG and DG6P, Chi and coworkers and Akabayashi and coworkers developed enzymatic assays (Akabayashi et al, 1989;Chi et al, 1978;Chi et al, 1993;Chi et al, 1987). Their methods are based on the HK-G6PDH assay described above and either directly detect NADPH derived from the reactions or detect NADPH by amplification with NADP cycling reagents.…”

Section: Methods Based On Fluorescent Glucose Analogs 2 or 6-nbdg (Flumentioning

confidence: 99%

Evaluation Methods for Facilitative Glucose Transport in Cells and Their Applications

Yamamoto¹,

Ashida

2012

FSTR

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Glucose is used as an energy source in most organisms, from bacteria to humans. The facilitative glucose transport systems are ubiquitous in living cells and are responsible for the movement of glucose across the cell membrane. Since transport of glucose is essential for life and is carefully regulated, glucose uptake assays are performed in a vast number of biological studies. Previously, the method of choice employed radio-labeled hexose derivatives, but now researchers can select several non-radioisotopic methods to evaluate glucose uptake. In the present review, we introduce the various methodologies used to conduct glucose uptake assays and evaluate their application.

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“…Measurement of 2DG and 2DG-6-P can be achieved by a number of various techniques, such as NMR (6), enzymatic assays (7)(8)(9), and scintillation counting of radiolabeled metabolites (2,(10)(11)(12)(13)(14). While each of the foregoing methods has advantages, notable drawbacks include the requirement for sophisticated instrumentation and expertise, lack of sensitivity, and logistical and administrative constraints, respectively.…”

Section: Introductionmentioning

confidence: 99%

A Fast and Sensitive Method Combining Reversed-Phase Chromatography with High Resolution Mass Spectrometry to Quantify 2-Fluoro-2-Deoxyglucose and Its Phosphorylated Metabolite for Determining Glucose Uptake

Aw¹,

Muoio²,

Zhang³

2019

Preprint

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Quantative measurements of the glucose analogue, 2-deoxyglucose (2DG), and its phosphorylated metabolite (2-deoxyglucose-6-phosphate (2DG-6-P)) are critical for the measurement of glucose uptake. While the field has long identified the need for sensitive and reliable assays that deploy non-radiolabled glucose analogues to assess glucose uptake, no analytical MS-based methods exist to detect trace amounts in complex biological samples. In the present work, we show that 2DG is poorly suited for MS-based methods due to interfering metabolites. We therefore developed and validated an alternative C18-based LC-Q-Exactive-Orbitrap-MS method using 2-fluoro-2-deoxyglucose (2FDG) to quantify both 2FDG and 2FDG-6-P by measuring the sodium adduct of 2FDG in the positive mode and deprotonation of 2FDG-6-P in the negative mode. The low detection limit of this method can reach 81.4 and 48.8 fmol for both 2FDG and 2FDG-6-P, respectively. The newly developed method was fully validated via calibration curves in the presence and absence of biological matrix. The present work is the first successful LC-MS method that can quantify trace amounts of a nonradiolabeled glucose analogue and its phosphorylated metabolite and is a promising analytical method to determine glucose uptake in biological samples.

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A Refinement of the Akabayashi-Saito-Kato Modification of the Enzymatic Methods for 2-Deoxyglucose and 2-Deoxyglucose 6-Phosphate

Cited by 10 publications

References 0 publications

Ras signaling in the activation of glucose transport by insulin.

Ras signaling in the activation of glucose transport by insulin.

Evaluation Methods for Facilitative Glucose Transport in Cells and Their Applications

A Fast and Sensitive Method Combining Reversed-Phase Chromatography with High Resolution Mass Spectrometry to Quantify 2-Fluoro-2-Deoxyglucose and Its Phosphorylated Metabolite for Determining Glucose Uptake

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