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“Fluorescent glycogen” formation with sensibility for in vivo and in vitro detection

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Abstract

There are presently many methods of detecting complex carbohydrates, and particularly glycogen. However most of them require radioisotopes or destruction of the tissue and hydrolysis of glycogen to glucose. Here we present a new method based on the incorporation of 2-NBDG (2-{N-[7-nitrobenz-2-oxa-1, 3-diazol 4-yl] amino}-2-deoxyglucose), a d-glucose fluorescent derivative, into glycogen. Two kinds of approaches were carried out by using Clone 9 rat hepatocytes as a cellular model; (1) Incubation of cell lysates with 2-NBDG, carbohydrate precipitation in filters and measurement of fluorescence in a microplate reader (2) Incubation of living hepatocytes with 2-NBDG and recording of fluorescence images by confocal microscopy. 2-NBDG labeled glycogen in both approaches. We confirmed this fact by comparison to the labeling obtained with a specific monoclonal anti-glycogen antibody. Also drugs that trigger glycogen synthesis or degradation induced an increase or decrease of fluorescence, respectively. This is a simple but efficient method of detecting glycogen with 2-NBDG. It could be used to record changes in glycogen stores in living cells and cell-free systems and opens the prospect of understanding the role of this important energy reserve under various physiological and pathophysiological conditions.

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Acknowledgements

This work was funded with grants from the following agencies:

Ministerio de Ciencia y Tecnología, Spain; Grant Number: SAF2003-08765-C03-02, REN2001-2959-C04-03, REN2003-06598-C02-01, AGL2004-08268-02-O2/ALI. Xunta de Galicia, Spain; Grant Number: PGIDT99INN26101, PGIDIT03AL26101PR and PGIDIT04TAL261005PR.

Fondo de Investigaciones Sanitarias, Spain; Grant Number: FISS REMA-G03-007. EU VIth Frame Program; Grant Number: IP FOOD-CT-2004-06988 (BIOCOP) and STREP FOOD-CT-2004-514055 (DETECTOX), CRP 030270-2 (SPIES-DETOX).

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Authors and Affiliations

  1. Departamento de Farmacologia, Facultad de Veterinaria de Lugo, Universidad de Santiago de Compostela, 27002, Lugo, Spain

    M. Carmen Louzao, Begoña Espiña & Luis M. Botana

  2. Departamento de Fisiologia Animal, Facultad de Veterinaria de Lugo, Universidad de Santiago de Compostela, 27002, Lugo, Spain

    Mercedes R. Vieytes, Felix V. Vega & Juan A. Rubiolo

  3. Biostructural Science, Tokyo Medical & Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8549, Japan

    Otto Baba & Tatsuo Terashima

Authors
  1. M. Carmen Louzao
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  2. Begoña Espiña
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  3. Mercedes R. Vieytes
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  4. Felix V. Vega
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  8. Luis M. Botana
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Correspondence to Luis M. Botana.

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Louzao, M.C., Espiña, B., Vieytes, M.R. et al. “Fluorescent glycogen” formation with sensibility for in vivo and in vitro detection. Glycoconj J 25, 503–510 (2008). https://doi.org/10.1007/s10719-007-9075-7

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  • DOI: https://doi.org/10.1007/s10719-007-9075-7

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