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

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.

Defects in calcium homeostasis and mitochondria can be reversed in Pompe disease

“…32 Lysosomal glycogen was evaluated by the incorporation of 2-NBDG (Life Technologies, N13195), a D-glucose fluorescent derivative (2-deoxyglucose), into glycogen as described. 85 Human primary muscle cells were provided by Dr. Benedikt Schoser (Friedrich-Baur-Institute, Ludwig-Maximillian University, Munich, Germany). Three human samples were used: one was from a healthy individual (# 94/13) and 2 from patients with adult form of Pompe disease (# FBI-542-2004, mutations c. In addition, we made a new in vitro model of Pompe disease-immortalized KO muscle cells, in which lysosomes are labeled with mCherry-LAMP1.…”

Disruption of the Actin Cytoskeleton Induces Fluorescent Glucose Accumulation on the Rat Hepatocytes Clone 9

“…Once this dye is inside the cells, it can follow the D-glucose degradation route or the glycogen formation pathway. In a previous work, we set up a method by using 2-NBDG that allows a noninvasive detection of glycogen being also a rapid testing of drugs that modify the incorporation or release of glucose from glycogen [19].…”

“…Any failure in this metabolic pathway frequently entails severe disorders in the organism. Recent investigations discovered that 2-NBDG (2-{N-[7-nitrobenz-2-oxa-1, 3-diazol 4-yl] amino}-2-deoxyglucose), a fluorescent analogue of the D-glucose that gets into cells by using the same transporters [14][15][16][17][18], could be used to monitor the rate of glycogen synthesis in cells [19]. D-glucose uptake by cells and the actin cytoskeleton have been previously related.…”

Production of Functionally Active Palytoxin-like Compounds by Mediterranean <i>Ostreopsis cf. siamensis</i>

“…Cells were cultured in a nutrient mixture F-12 Ham Kaighn's modification supplemented with 2.5 g/L NaHCO 3 , 28 mg/L streptomycin sulfate salt, 17 mg/L penicillin G potassium salt and 10% FBS. Cells were grown on 60 mm tissue culture plates in a humidified atmosphere with 5% CO 2 at 37°C, and subcultured by transferring cells released by the application of 0.25% trypsin-EDTA [54].…”