Name: biochemical titration of glycogen in vitro
Uploaded: 2013-11-24T19:00:00
Description: Watch this Scientific Journal Video about Biochemical Titration of Glycogen In vitro at JoVE.com

We are grateful to the Dr. Thierry Pourcher for allowing us to use the fluorescence spectrometer and for his help. The laboratory is funded by the Ligue Nationale Contre le Cancer (&#233;quipe labellis&#233;e), the Association pour la Recherche contre le Cancer, the Institut National du Cancer (INCa), the Agence Nationale pour la Recherche, METOXIA (EU program FP7), the Centre A. Lacassagne, the Centre National de la Recherche Scientifique, the Institut National de la Sant&#233; et de la Recherche M&#233;dicale and the University of Nice (<a href="http://www.unice.fr/isdbc/" target="_blank">http://www.unice.fr/isdbc/</a>). We thank Dr. M Christiane Brahimi-Horn for critical reading and editorial correction.

Biochemical Titration of Glycogen
1. Cell Lysis
<ol>
	<li>Seed cells at a concentration of 0.5-2 x 106 per 100 mm diameter dish.</li>
	<li>Treatment: incubate cells 24, 48, or 72 hr in low oxygen concentration (hypoxia) in a Bug-Box anaerobic work station (Ruskinn Technology Biotrace International Plc, Bridgend, UK) set at 1% or 0.1% O2, 94% or 94.9% N2, and 5% CO2. In parallel, incubate cells in normoxia (21% O2, 5% CO2). Chang...

<ol>
	<li>Pelletier, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+Synthesis+is+Induced+in+Hypoxia+by+the+Hypoxia-Inducible+Factor+and+Promotes+Cancer+Cell+Survival.">Glycogen Synthesis is Induced in Hypoxia by the Hypoxia-Inducible Factor and Promotes Cancer Cell Survival.</a> Front Oncol. 2, (2012).</li><li>Alonso, M. D., Lomako, J., Lomako, W. M., Whelan, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+look+at+the+biogenesis+of+glycogen.">A new look at the biogenesis of glycogen.</a> FASEB J. 9, 1126-1137 (1995).</li><li>Bollen, M., Keppens, S., Stalmans, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+features+of+glycogen+metabolism+in+the+liver.">Specific features of glycogen metabolism in the liver.</a> Biochem. J. 336 (Pt 1), 19-31 (1998).</li><li>Parker, G. J., Koay, A., Gilbert-Wilson, R., Waddington, L. J., Stapleton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMP-activated+protein+kinase+does+not+associate+with+glycogen+alpha-particles+from+rat+liver.">AMP-activated protein kinase does not associate with glycogen alpha-particles from rat liver.</a> Biochem. Biophys. Res. Commun. 362, 811-815 (2007).</li><li>Sullivan, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature+of+alpha+and+beta+particles+in+glycogen+using+molecular+size+distributions.">Nature of alpha and beta particles in glycogen using molecular size distributions.</a> Biomacromolecules. 11, 1094-1100 (2010).</li><li>Chee, N. P., Geddes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+liver+glycogen.">The structure of liver glycogen.</a> FEBS Lett. 73, 164-166 (1977).</li><li>Roach, P. J., Depaoli-Roach, A. A., Hurley, T. D., Tagliabracci, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+and+its+metabolism:+some+new+developments+and+old+themes.">Glycogen and its metabolism: some new developments and old themes.</a> Biochem. J. 441, 763-787 (2012).</li><li>Moses, S. W., Bashan, N., Gutman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+metabolism+in+the+normal+red+blood+cell.">Glycogen metabolism in the normal red blood cell.</a> Blood. 40, 836-843 (1972).</li><li>Agbanyo, M., Taylor, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+3-deoxy-3-fluoro-D-glucose+into+glycogen+and+trehalose+in+fat+body+and+flight+muscle+in+Locusta+migratoria.">Incorporation of 3-deoxy-3-fluoro-D-glucose into glycogen and trehalose in fat body and flight muscle in Locusta migratoria.</a> Biosci. Rep. 6, 309-316 (1986).</li><li>Louzao, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Fluorescent+glycogen"+formation+with+sensibility+for+in+vivo+and+in+vitro+detection.">"Fluorescent glycogen" formation with sensibility for in vivo and in vitro detection.</a> Glycoconj. J. 25, 503-510 (2008).</li><li>Sheehan, D. C. H., B, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Theory and practice of histotechnology. , (1980).</li><li>Baba, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+monoclonal+antibody+that+recognizes+glycogen+and+its+application+for+immunohistochemistry.">Production of monoclonal antibody that recognizes glycogen and its application for immunohistochemistry.</a> Kokubyo Gakkai Zasshi. 60, 264-287 (1993).</li></ol>

Biochemical titration of glycogen in vitro allows an accurate quantification of cells glycogen content. Comparing to some other techniques (PAS, immunofluorescence with a glycogen antibody, etc.), this titration is very specific, sensitive and reproducible. Moreover, the method is convenient since it requires no radioactivity but a fluorescence spectrometer. However, this technique is purely quantitative and does not provide information about glycogen distribution in the cell.
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Glycogen is a multibranched polymer of glucose residues, which is present in the cytoplasm of many cell types. It is one of the main forms of energy storage in cells and plays an important role in glucose metabolism. Most mammalian cells are able to produce and store glycogen, which can be rapidly degraded into glucose to promote glycolysis and ATP production during metabolic stress. Hepatocytes produce massive amounts of glycogen to regulate the blood sugar level thereby providing a continuous supply of glucose to the body. In contrast, the concentration of glycogen in other cells (muscles, red blood cells,&#160;etc.) is relatively low. However, locally, these quantities are sufficient to provide energy in the&#160;short-term when the cells are suddenly exposed to an environment deprived of nutrients.
Glycogen synthesis and glycogen breakdown follows the same steps in all tissues (Figure 1). Firstly, glucose enters cells by glucose transporters (GLUTs), and is rapidly converted from glucose-6-phosphate (G6P) into glucose-1-phosphate (G1P) by phosphoglucomutase. G1P is then modified into UDP-glucose, and the carbon C1 of UDP-glucose is attached to a tyrosine residue of glycogenin, the anchoring protein of glycogen. This molecule, considered a glycogen primer, is extended by attachment of the UDP-glucose to the terminal glucose by an &#945;(1&#8594;4) bond via the glycogen synthase. Finally, when a linear chain of over 11 glucose residues is formed, the branching enzyme transfers a terminal oligosaccharide constituted by a minimum of 6 glucose residues to another glucose of the chain nearby through an &#945;(1&#8594;6) linkage. The repetition of this process gives a massive fractal structure containing branches that form a helix with 6.5 glucose per turn. Glycogen can be reversely hydrolyzed to glucose by the concerted action of debranching enzymes that hydrolyze the &#945;(1&#8594;6) bound and glycogen phosphorylase that hydrolyzes the &#945;(1&#8594;4) glycosidic bound between the last residue of glucose of a branch and the glycogen molecule. This reaction called glycogenolysis is activated by increasing levels of AMP (reflecting ATP consumption), and inhibited by glucose and ATP2,3.
By electron microscopy, glycogen molecules have been described in many cell types as &#946; free particles (or glycogen monoparticles) of 15-30 nm in diameter. In specific cell types such as hepatocytes, &#946; particles can be assembled into a complex to form rosettes, also known as &#945; particles that vary in diameter from 80 nm to a maximum of 200 nm4. The way these &#946; particles are bonded to form larger clusters of &#945; particles is still not fully elucidated. Some evidence tends to prove that &#946; particles can be bonded by covalent bonding5, hydrogen bonding, or even through protein-protein interactions6. The amount of glycogen stored in the cells depends on many parameters: (I) the amount of glycogenin in the cell that initiates glycogen synthesis; (II) the activity of glycogen synthase and phosphorylase regulated by protein phosphorylation/dephosphorylation; (III) the concentration of glucose in the cells, which is dependent on several parameters such as the supply of glucose from the vascular system and glucose uptake by cells. Glycogen stores are tightly regulated by allosteric regulation of biosynthetic hormones through intermediate metabolites, by hormones regulating energy metabolism, and by nutrient sensing signaling pathways7.
It is important to be able to quantify glycogen in biological samples to better understand the importance of glycogen metabolism at the whole body and cellular level. We describe here a precise, reproducible and convenient biochemical in vitro assay for glycogen. This technique is based on the quantification glucose fluorescence before and after specific hydrolysis of glycogen.
Other methods exist to estimate the level of glycogen in the cells, but most of them are not quantitative. One of the first techniques described for quantification of glycogen in cells was based on measurement of [14C]-glucose incorporation into glycogen8,9. The use of radioactivity makes this process more difficult to handle but it has the advantage of providing the rate of glucose incorporation into glycogen and in distinguishing between the distribution of glucose residues on the outer branches and in the core of the molecule (it also requires an additional &#946;-amylolysis and a chromatographic step). Another technique was developed more recently and is based on the incorporation of 2-NBDG (2-{N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl] amino}-2-deoxyglucose), a 2-deoxyglucose fluorescent derivative, into glycogen10. The measured fluorescence intensity reflects the amount of glycogen produced and can be measured with a fluorescence reader. Distribution of fluorescence in the cell can also be evaluated by confocal microscopy.
Among the other nonquantitative techniques, Periodic Acid-Schiff staining (PAS) is perhaps the most common. It can be used for the detection of glycogen in fixed cells, tissue sections in paraffin or frozen. This histological technique colors non specifically polysaccharides, glycolipids, glycoproteins, cellulose and neutral mucins in purple. The specificity of this test can be increased by treatment of fixed cells or tissue sections with diastase, which specifically digests glycogen. Thereafter, the level of glycogen can be qualitatively estimated by comparing unhydrolyzed samples (all carbohydrate modified macromolecules) to hydrolyzed samples (carbohydrate modified macromolecules except glycogen). PAS staining and microscopic analysis, unlike biochemical assays of glycogen, provides information concerning the distribution of glycogen in the cell, which can be diffused or concentrated in a certain part of the cell. However, even though PAS staining estimates differences in glycogen accumulation between different conditions, it is not quantitative11.
A monoclonal mouse antibody originally made using mandibular condylar cartilage as the antigen has been shown to react with glycogen in cells and with purified glycogen in vitro12. As this antibody specifically recognizes glycogen-related sugar chains, it is a useful tool for the detection of glycogen by immunohistochemistry in a more specific way than the PAS staining.
Electron microscopy is another technique that allows visualization of grains of glycogen in cells and evaluation of the degree of glycogen storage. In fact, glycogen &#946; particles are easily recognizable with an electron microscopy as electron dense granules1.

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			<td>REAGENTS</td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>31966.047</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Glycogen Assay Kit</td>
			<td>Abcam</td>
			<td>ab65620 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td></td>
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 <td></td>
 <td>EQUIPMENT</td>
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			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments (optional)</td>
		 </tr>
		 <tr>
			<td>Fluorescence Spectrometer</td>
			<td>PerkinElmer</td>
			<td>LS 50B</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

biochemical titration of glycogen in vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

A low level of oxygen (hypoxia) in tumors signals to tumor cells the need to store energy to handle subsequent nutrient depletion, so as to survive. As glycogen is the main energetic polymer of glucose in mammalian cells, we studied the regulation of glycogen storage in hypoxia. The calculation and standardization of the concentration of glycogen in cell lysates must be performed on the raw data of fluorescence as shown in Tables 1, 2, and 3. The biochemical assay for gl...

Watch this Scientific Journal Video about Biochemical Titration of Glycogen In vitro at JoVE.com

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

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