"Fluorescent glycogen" formation with sensibility for in vivo and in vitro detection.
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.
Louzao
M Carmen
MC
Departamento de Farmacologia, Facultad de Veterinaria de Lugo, Universidad de Santiago de Compostela, 27002 Lugo, Spain. ffmclooj@lugo.usc.es
Espiña
Begoña
B
Vieytes
Mercedes R
MR
Vega
Felix V
FV
Rubiolo
Juan A
JA
Baba
Otto
O
Terashima
Tatsuo
T
Botana
Luis M
LM
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D002460:Cell Line; D002474:Cell-Free System; D003847:Deoxyglucose / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D005456:Fluorescent Dyes / Q000032:analysis* / Q000737:chemistry; D006003:Glycogen / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D022781:Hepatocytes / Q000378:metabolism; D007075:Immunoglobulin M / Q000737:chemistry / Q000276:immunology; D018613:Microscopy, Confocal; D015394:Molecular Structure; D051381:Rats
D000906:Antibodies; D005456:Fluorescent Dyes; D007075:Immunoglobulin M; D006003:Glycogen; D003847:Deoxyglucose; D009327:4-Chloro-7-nitrobenzofurazan; C098340:2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose