The author would like to thank Karoline Dittmer and Andrew Brittingham for their insights and many helpful discussions. This work was supported in part by grants from the Iowa Osteopathic Education and Research Fund (IOER 03-17-05 and 03-20-04).

<ol>
	<li>Wilson, W. 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=Regulation+of+glycogen+metabolism+in+yeast+and+bacteria.">Regulation of glycogen metabolism in yeast and bacteria.</a> FEMS Microbiology Reviews. 34 (6), 952-985 (2010).</li><li>Ralton, J. E., Sernee, M. F., McConville, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+and+function+of+carbohydrate+reserve+biosynthesis+in+parasitic+protists.">Evolution and function of carbohydrate reserve biosynthesis in parasitic protists.</a> Trends in Parasitology. 1471 (21), 00144-00146 (2021).</li><li>Roach, P. J. <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.">Glycogen and its metabolism.</a> Current Molecular Medicine. 2 (2), 101-120 (2002).</li><li>Thomas, J. A., Schlender, K. K., Larner, 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+rapid+filter+paper+assay+for+UDPglucose-glycogen+glucosyltransferase,+including+an+improved+biosynthesis+of+UDP-14C-glucose.">A rapid filter paper assay for UDPglucose-glycogen glucosyltransferase, including an improved biosynthesis of UDP-14C-glucose.</a> Analytical Biochemistry. 25 (1), 486-499 (1968).</li><li>Fox, J., Kawaguchi, K., Greenberg, E., Preiss, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosynthesis+of+bacterial+glycogen.+Purification+and+properties+of+the+Escherichia+coli+B+ADPglucose:1,4-alpha-D-glucan+4-alpha-glucosyltransferase.">Biosynthesis of bacterial glycogen. Purification and properties of the Escherichia coli B ADPglucose:1,4-alpha-D-glucan 4-alpha-glucosyltransferase.</a> Biochemistry. 15 (4), 849-857 (1976).</li><li>Gilboe, D. P., Larson, K. L., Nuttall, F. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radioactive+method+for+the+assay+of+glycogen+phosphorylases.">Radioactive method for the assay of glycogen phosphorylases.</a> Analytical Biochemistry. 47 (1), 20-27 (1972).</li><li>Brown, D. H., Illingworth, B., Cori, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+the+de+novo+synthesis+of+polysaccharide+by+phosphorylase.">The mechanism of the de novo synthesis of polysaccharide by phosphorylase.</a> Proceedings of the National Academy of Sciences of the United States of America. 47 (4), 479-485 (1961).</li><li>Nelson, T. E., Larner, 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+rapid+micro+assay+method+for+amylo-1,6-glucosidase.">A rapid micro assay method for amylo-1,6-glucosidase.</a> Analytical Biochemistry. 33 (1), 87-101 (1970).</li><li>Leloir, L. F., Olavarria, J. M., Goldemberg, S. H., Carminatti, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosynthesis+of+glycogen+from+uridine+diphosphate+glucose.">Biosynthesis of glycogen from uridine diphosphate glucose.</a> Archives of Biochemistry and Biophysics. 81 (2), 508-520 (1959).</li><li>Danforth, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+synthetase+activity+in+skeletal+muscle.+Interconversion+of+two+forms+and+control+of+glycogen+synthesis.">Glycogen synthetase activity in skeletal muscle. Interconversion of two forms and control of glycogen synthesis.</a> Journal of Biological Chemistry. 240, 588-593 (1965).</li><li>Wayllace, N. Z., 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=An+enzyme-coupled+continuous+spectrophotometric+assay+for+glycogen+synthases.">An enzyme-coupled continuous spectrophotometric assay for glycogen synthases.</a> Molecular Biology Reports. 39 (1), 585-591 (2012).</li><li>Shapiro, B., Wertheimer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorolysis+and+synthesis+of+glycogen+in+animal+tissues.">Phosphorolysis and synthesis of glycogen in animal tissues.</a> Biochemical Journal. 37 (3), 397-403 (1943).</li><li>Mezl, V. A., Knox, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+two+methods+for+the+assay+of+glycogen+phosphorylase+in+tissue+homogenates.">Comparison of two methods for the assay of glycogen phosphorylase in tissue homogenates.</a> Enzyme. 13 (4), 197-202 (1972).</li><li>Mendicino, J., Afou-Issa, H., Medicus, R., Kratowich, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fructose-1,+6-diphosphatase,+phosphofructokinase,+glycogen+synthetase,+phosphorylase,+and+protein+kinase+from+swine+kidney.">Fructose-1, 6-diphosphatase, phosphofructokinase, glycogen synthetase, phosphorylase, and protein kinase from swine kidney.</a> Methods in Enzymology. 42, 375-397 (1975).</li><li>Schreiber, W. E., Bowling, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+assay+of+glycogen+phosphorylase+in+the+direction+of+phosphorolysis.">An automated assay of glycogen phosphorylase in the direction of phosphorolysis.</a> Annals of Clinical Biochemistry. 27, 129-132 (1990).</li><li>Nelson, T. E., Kolb, E., Larner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+properties+of+rabbit+muscle+amylo-1,6-glucosidase-oligo-1,4-1,4-transferase.">Purification and properties of rabbit muscle amylo-1,6-glucosidase-oligo-1,4-1,4-transferase.</a> Biochemistry. 8 (4), 1419-1428 (1969).</li><li>Taylor, C., Cox, A. J., Kernohan, J. C., Cohen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Debranching+enzyme+from+rabbit+skeletal+muscle.+Purification,+properties+and+physiological+role.">Debranching enzyme from rabbit skeletal muscle. Purification, properties and physiological role.</a> European Journal of Biochemistry. 51 (1), 105-115 (1975).</li><li>Makino, Y., Omichi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+glycogen+debranching+enzyme+from+porcine+brain:+evidence+for+glycogen+catabolism+in+the+brain.">Purification of glycogen debranching enzyme from porcine brain: evidence for glycogen catabolism in the brain.</a> Bioscience, Biotechnology, and Biochemistry. 70 (4), 907-915 (2006).</li><li>Lee, E. Y. C., Whelan, W. J., Boyer, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Enzymes Vol. 5. , 191-234 (1971).</li><li>Yu, X., Houtman, C., Atalla, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complex+of+amylose+and+iodine.">The complex of amylose and iodine.</a> Carbohydrate Research. 292, 129-141 (1996).</li><li>Boyer, C., Preiss, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosynthesis+of+bacterial+glycogen.+Purification+and+properties+of+the+Escherichia+coli+b+alpha-1,4,-glucan:+alpha-1,4-glucan+6-glycosyltansferase.">Biosynthesis of bacterial glycogen. Purification and properties of the Escherichia coli b alpha-1,4,-glucan: alpha-1,4-glucan 6-glycosyltansferase.</a> Biochemistry. 16 (16), 3693-3699 (1977).</li><li>Krisman, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+colorimetric+estimation+of+glycogen+with+iodine.">A method for the colorimetric estimation of glycogen with iodine.</a> Analytical Biochemistry. 4, 17-23 (1962).</li><li>Friedman, D. L., Larner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+UDPG-alpha-glucan+transglucosylase.+iii.+Interconversion+of+two+forms+of+muscle+UDPG-alpha-glucan+transglucosylase+by+a+phosphorylation-dephosphorylation+reaction+sequence.">Studies on UDPG-alpha-glucan transglucosylase. iii. Interconversion of two forms of muscle UDPG-alpha-glucan transglucosylase by a phosphorylation-dephosphorylation reaction sequence.</a> Biochemistry. 2, 669-675 (1963).</li><li>Hanashiro, I., Roach, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+of+muscle+glycogen+synthase+that+disable+activation+by+glucose+6-phosphate.">Mutations of muscle glycogen synthase that disable activation by glucose 6-phosphate.</a> Archives of Biochemistry and Biophysics. 397 (2), 286-292 (2002).</li><li>Huang, K. P., Cabib, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+glycogen+synthetase+in+the+glucose+6-phosphate-dependent+form.+I.+Purification+and+properties.">Yeast glycogen synthetase in the glucose 6-phosphate-dependent form. I. Purification and properties.</a> Journal of Biological Chemistry. 249 (12), 3851-3857 (1974).</li><li>Pederson, B. A., Cheng, C., Wilson, W. A., Roach, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+glycogen+synthase.+Identification+of+residues+involved+in+regulation+by+the+allosteric+ligand+glucose-6-P+and+by+phosphorylation.">Regulation of glycogen synthase. Identification of residues involved in regulation by the allosteric ligand glucose-6-P and by phosphorylation.</a> Journal of Biological Chemistry. 275 (36), 27753-27761 (2000).</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> Biochemical Journal. 441 (3), 763-787 (2012).</li><li>Gosselin, S., Alhussaini, M., Streiff, M. B., Takabayashi, K., Palcic, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+continuous+spectrophotometric+assay+for+glycosyltransferases.">A continuous spectrophotometric assay for glycosyltransferases.</a> Analytical Biochemistry. 220 (1), 92-97 (1994).</li><li>Wilson, W. A., Pradhan, P., Madhan, N., Gist, G. C., Brittingham, 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+synthase+from+the+parabasalian+parasite+Trichomonas+vaginalis:+An+unusual+member+of+the+starch/glycogen+synthase+family.">Glycogen synthase from the parabasalian parasite Trichomonas vaginalis: An unusual member of the starch/glycogen synthase family.</a> Biochimie. 138, 90-101 (2017).</li><li>Krisman, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=alpha-1,4-glucan:+alpha-1,4-glucan+6-glycosyltransferase+from+liver.">alpha-1,4-glucan: alpha-1,4-glucan 6-glycosyltransferase from liver.</a> Biochimica et Biophysica Acta. 65, 307-315 (1962).</li><li>Sandhya Rani, M. R., Shibanuma, K., Hizukuri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fine+structure+of+oyster+glucogen.">The fine structure of oyster glucogen.</a> Carbohydrate Research. 227, 183-194 (1992).</li><li>Dittmer, K. E., Pradhan, P., Tompkins, Q. C., Brittingham, A., Wilson, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+characterization+of+glycogen+branching+and+debranching+enzymes+from+the+parasitic+protist+Trichomonas+vaginalis.">Cloning and characterization of glycogen branching and debranching enzymes from the parasitic protist Trichomonas vaginalis.</a> Biochimie. 186, 59-72 (2021).</li></ol>

There are no known conflicts of interest associated with this work and there has been no financial support for this work that could have influenced its outcome.

In general, the key advantages of all of the methods presented are their low cost, ease, speed, and lack of reliance upon specialized equipment. The major disadvantage that they all share is sensitivity compared to other available methods. The sensitivity of the procedures that involve production or consumption of NADH/NADPH are easy to estimate. Given that the extinction coefficient of NADH/NADPH is 6.22 M-1 cm-1, simple arithmetic indicates that ~10-20 &#181;M changes in concentration can be readily detected. With the assay volumes described in the current article, this corresponds to the ability to measure quantities in the range of ~10-20 nmol. Arguably, this is fairly sensitive. However, it is possible to adjust the specific activity of radiolabeled substrates such that sensitivity can be increased beyond the limit of the spectrophotometric assays quite readily. Although the branching enzyme assay and the qualitative assessment of glycogen branching both rely upon the formation of complexes between iodine and &#945;1,4-linked glucose polymers, the output from each assay is different and their sensitivities are likewise different. Specific considerations for each of the assays described are discussed below.
Determination of glycogen synthase activity 
The coupled enzyme assay described here allows for continuous assay of glycogen synthase activity, which is useful in the determination of kinetic parameters. It is also readily scalable and a very similar procedure has been described for use in microtiter plates, allowing for highly parallel measurements of enzyme activity to be made28. We routinely use this assay with purified, recombinant glycogen synthases29. However, the procedures described were originally developed for use in tissue homogenates (for examples, see Danforth10 and Leloir et al.9). A caveat here is that the tissue homogenate must be appropriately diluted prior to use. The dilution is necessary to reduce the turbidity of the homogenate and interference from competing enzymes/substrates in the homogenate. Furthermore, to account for NADH consumption that is not dependent upon glycogen synthase activity, blank reactions that contain all reaction components except UDP-glucose should be included. The glycogen synthase activity is then determined by subtracting the rate of change in NADH absorbance in the absence of UDP-glucose from that obtained in the presence of this compound.
Determination of glycogen phosphorylase activity 
Like the glycogen synthase assay, the spectrophotometric measurement of phosphorylase activity can be performed in a continuous manner, while other phosphorylases assays are stopped assays13. It is also readily adaptable for use in microtiter plates or other high-throughput applications15. Again, its use with tissue homogenates requires appropriate dilution to reduce turbidity/interfering reactions. For example, glucose-6-phosphate is a rather abundant metabolite and its presence in a tissue homogenate will result in NADPH production via the glucose-6-phosphate dehydrogenase coupling enzyme independent of phosphorylase activity. When working with tissue homogenates, control reactions that contain appropriately diluted tissue homogenate and all other assay components except phosphate and glycogen should be included. Phosphorylase activity is then calculated by subtracting the NADPH production in the absence of glycogen/phosphate from that which occurs in the presence of these compounds. Specific recommendations relating to the appropriate degree of dilution of various types of mammalian tissue extract can be found in Mezl et al.13.
Determination of glycogen debranching enzyme activity 
Although described here as a stopped assay, this procedure can be readily adapted and performed in a continuous fashion16. Since the assay relies upon the production of glucose, its use in crude tissue extracts must consider the release of glucose from phosphorylase limit dextrin by enzymes other than debranching enzyme, and the generation of glucose by the action of such enzymes on endogenous glycogen present in the tissue extract. The question of endogenous glycogen is easily addressed with a control reaction to which no phosphorylase limit dextrin is added. The presence of other &#945;-glucosidase activities can be estimated in parallel reactions where maltose, rather than phosphorylase limit dextrin, is present as a substrate.
Determination of glycogen branching enzyme activity 
Of the various quantitative assays discussed, the colorimetric branching enzyme assay is by far the least sensitive. Indeed, it has been estimated that the sensitivity is around 50-100 fold less than that achieved with the radiochemical method, where stimulation of the glycogen phosphorylase reaction by the addition of branching enzyme is measured21. Similar to the debranching enzyme assay, the branching enzyme assay is also sensitive to the presence of contaminating glucosidase activities, since digestion of the amylose will impact iodine binding. Some workarounds have been proposed to allow the use of assays similar to that described here in the presence of contaminating glucosidase activities30. However, in our view this assay is best suited to the study of purified or partially purified glycogen branching enzymes, where such interfering activities are minimal or absent.
Qualitative assessment of the extent of glycogen branching 
The detailed analysis of glycogen branching is rather laborious, typically involving a combination of enzymatic digestion, chemical modification, and a variety of separation techniques to analyze the products generated31. While the colorimetric assay described here clearly cannot yield comparable information on the fine structure of glycogen, it does provide a simple and rapid measure of more or less branching. Furthermore, the spectra obtained do contain some additional information. For example, as discussed under Representative Results, samples of glycogen are typically present with absorbance peaks at ~400 nm and ~460 nm. The peak at ~400 nm apparently represents short outer chains in glycogen particles, since it is enhanced in glycogen isolated from yeast mutants lacking branching enzyme relative to wild type yeast32.

Glycogen is widely distributed in nature, with the compound being found in bacteria, many protists, fungi, and animals. In microorganisms, glycogen is important for cell survival when nutrients are limiting and, in higher organisms such as mammals, synthesis and degradation of glycogen serve to buffer blood glucose levels1,2,3. The study of glycogen metabolism is, therefore, of importance to such diverse fields as microbiology and mammalian physiology. Understanding glycogen metabolism requires studying the key enzymes of glycogen synthesis (glycogen synthase and the branching enzyme) and glycogen degradation (glycogen phosphorylase and debranching enzyme). The gold standard assays of glycogen synthase, phosphorylase, branching, and debranching enzyme activities employ radioactive isotopes. For example, glycogen synthase is generally measured in a stopped radiochemical assay by following the incorporation of glucose from UDP-[14C]glucose (in the case of animal and fungal enzymes) or ADP-[14C]glucose (in the case of bacterial enzymes) into glycogen4,5. Similarly, glycogen phosphorylase is measured in the direction of glycogen synthesis, following the incorporation of glucose from [14C]glucose-1-phosphate into glycogen6. The branching enzyme is assayed by measuring the ability of this enzyme to stimulate the incorporation of [14C]glucose from glucose-1-phosphate into &#945;1,4-linked chains by glycogen phosphorylase7, and debranching enzyme activity is determined by following the ability of the enzyme to incorporate [14C]glucose into glycogen8. While very sensitive, allowing their use in crude cell extracts with low levels of enzyme activity, the radioactive substrates are expensive and subject to the regulatory requirements attendant to radioisotope use. These barriers place the use of certain assays out of the reach of many workers. However, over the course of many years, an impressive variety of spectrophotometric approaches to the measurement of these enzymes have been described. In general, these approaches ultimately rely upon measuring the production or consumption of NADH/NADPH, or the generation of colored complexes between glycogen and iodine. Thus, they are straightforward and can be carried out using simple spectrophotometers equipped with only tungsten or xenon flash lamps.
Spectrophotometric assays of glycogen synthase rely upon measuring the nucleoside diphosphate released from the sugar nucleotide donor as glucose is added to the growing glycogen chain9,10. The procedure for measuring glycogen synthase activity described in section 1 of the protocol, below, is a modification of that outlined by Wayllace et al.11, and the coupling scheme is shown below:
(Glucose)n + UPD-glucose &#8594; (Glucose)n+1 + UDP
UDP + ATP &#8594; ADP + UTP
ADP + phosphoenolpyruvate &#8594; pyruvate + ATP
Pyruvate + NADH + H+ &#8594; Lactate + NAD+
Glycogen synthase adds glucose from UDP-glucose onto glycogen. The UDP generated in this process is converted to UTP by nucleoside diphosphate kinase (NDP kinase), in a reaction that generates ADP. The ADP, in turn, then serves as a substrate for pyruvate kinase, which phosphorylates the ADP using phosphoenolpyruvate as a phosphate donor. The resulting pyruvate is converted to lactate by the enzyme lactate dehydrogenase in a reaction that consumes NADH. The assay can, therefore, be performed in a continuous fashion, monitoring the decrease in absorbance at 340 nm as NADH is consumed. It is readily adapted for use with enzymes that require ADP-glucose as a glucose donor. Here, the coupling steps are simpler since the ADP released by the action of glycogen synthase is directly acted upon by pyruvate kinase.
There are a variety of spectrophotometric assays available for the determination of glycogen phosphorylase activity. In the classical version, the enzyme is driven backward, in the direction of glycogen synthesis, as shown below:
(Glucose)n + Glucose-1-phosphate &#8594; (Glucose)n+1 + Pi
At timed intervals, aliquots of the reaction mixture are removed, and the amount of phosphate liberated is quantified12,13. In our hands, this assay has been of limited use due to the presence of readily measurable free phosphate in many commercial preparations of glucose-1-phosphate, combined with the high concentrations of glucose-1-phosphate required for phosphorylase action. Rather, we have routinely employed an alternative assay that measures the glucose-1-phosphate released as glycogen is degraded by phosphorylase13. A coupled reaction scheme, illustrated below, is employed.
(Glucose)n + Pi &#8594; (Glucose)n-1 + Glucose-1-phosphate
Glucose-1-phosphate &#8594; Glucose-6-phosphate
Glucose-6-phosphate + NADP+ &#8594; 6-phosphogluconolactone + NADPH + H+
The glucose-1-phosphate is converted into glucose-6-phosphate by phosphoglucomutase, and the glucose-6-phosphate is then oxidized to 6-phosphogluconolactone, with the concomitant reduction of NADP+ to NADPH. The procedure detailed in section 2 of the protocol, below, is derived from methods described by Mendicino et al.14 and Schreiber &#38; Bowling15. The assay can be readily performed in a continuous fashion, with the increase in absorbance at 340 nm over time, allowing the determination of the reaction rate.
Spectrophotometric determination of debranching enzyme activity relies upon the measurement of the glucose released by the action of the enzyme on phosphorylase limit dextrin16. This compound is made by treating glycogen exhaustively with glycogen phosphorylase. Since glycogen phosphorylase action stops 4 glucose residues away from an &#945;1,6-branch point, the limit dextrin contains glycogen, the outer chains of which have been shortened to ~4 glucose residues. Preparation of phosphorylase limit dextrin is described here, using a procedure derived from those developed by Taylor et al.17 and Makino &#38; Omichi18.
Debranching is a two-step process. The 4-&#945;-glucanotransferase activity of the bifunctional debranching enzyme first transfers three glucose residues from the branch point to the nonreducing end of a nearby &#945;1,4-linked glucose chain. The single, &#945;1,6-linked glucose residue remaining at the branch point is then hydrolyzed by the &#945;1,6-glucosidase activity19. The assay is typically performed in a stopped fashion, the glucose released after a given time (or series of times) being measured in a coupled enzyme assay as shown below:
(Glucose)n &#8594; (Glucose)n-1 + Glucose
Glucose + ATP &#8594; Glucose-6-phosphate + ADP
Glucose-6-phosphate + NADP+ &#8594; 6-phosphogluconolactone + NADPH + H+
The determination of NADPH produced gives a measure of glucose production. The procedure outlined in section 3 of the protocol, below, is based upon one described by Nelson et al.16. Like the other methods that rely upon the consumption or generation of NADH/NADPH, the assay is quite sensitive. However, the presence of amylases or other glucosidases, which can also liberate free glucose from phosphorylase limit dextrin, will cause significant interference (see Discussion).
The colorimetric determination of branching enzyme activity relies upon the fact that &#945;1,4-linked chains of glucose adopt helical structures that bind to iodine, forming colored complexes20. The color of the complex formed depends upon the length of the &#945;1,4-linked chains. Thus, amylose, which consists of long, largely unbranched chains of &#945;1,4-linked glucose forms a deep blue complex with iodine. In contrast, glycogens, the outer chains of which are generally in the order of only 6 to 8 glucose residues long, form orange-red complexes. If a solution of amylose is incubated with branching enzyme, the introduction of branches into the amylose results in the generation of shorter &#945;1,4-linked glucose chains. Thus, the absorption maximum of the amylose/iodine complexes shifts toward shorter wavelengths. The procedure discussed here is derived from that detailed by Boyer &#38; Preiss21 and branching enzyme activity is quantified as a reduction in absorption of the amylose/iodine complex at 660 nm over time.
As should be readily apparent from the discussion above, the fact that the colors of the complexes formed between iodine and &#945;1,4-glucose chains vary with the chain length means that the absorbance spectra of glycogen/iodine complexes should vary with the degree of glycogen branching. This is indeed the case, and less-branched glycogens/glycogens with longer outer chains absorb light at a longer wavelength than glycogens that are more branched/have shorter outer chains. The iodine staining reaction can therefore be used to gain rapid, qualitative data on the degree of glycogen branching22. The orange-brown color forms when glycogen complexes with iodine is not particularly intense. However, color development can be enhanced by the inclusion of saturated calcium chloride solution22. This boosts the sensitivity of the method some 10-fold and allows ready analysis of microgram quantities of glycogen. The assay for the determination of branching described in section 4 of the protocol, below, is adapted from a procedure developed by Krisman22. It is conducted simply by combining the glycogen sample with iodine solution and calcium chloride in a cuvette and collecting the absorption spectrum from 330 nm to 800 nm. The absorbance maximum shifts toward longer wavelengths as the degree of branching decreases.
Collectively, the methods described here provide simple, reliable means of assessing the activities of the key enzymes of glycogen metabolism, and for obtaining qualitative data on the extent of glycogen branching.

<table><tbody><tr><td>Amylopectin (amylose free) from waxy corn</td><td>Fisher Scientific</td><td>A0456</td><td></td></tr><tr><td>Amylose</td><td>Biosynth Carbosynth</td><td>YA10257</td><td></td></tr><tr><td>ATP, disodium salt</td><td>MilliporeSigma</td><td>A3377</td><td></td></tr><tr><td>D-Glucose-1,6-bisphosphate, potassium salt</td><td>MilliporeSigma</td><td>G6893</td><td></td></tr><tr><td>D-glucose-6-phosphate, sodium salt</td><td>MilliporeSigma</td><td>G7879</td><td></td></tr><tr><td>Glucose-6-phosphate dehydrogenase, Grade I, from yeast</td><td>MilliporeSigma</td><td>10127655001</td><td></td></tr><tr><td>Glycogen, Type II from oyster</td><td>MilliporeSigma</td><td>G8751</td><td></td></tr><tr><td>Hexokinase</td><td>MilliporeSigma</td><td>11426362001</td><td></td></tr><tr><td>Methacrylate cuvettes, 1.5 mL</td><td>Fisher Scientific</td><td>14-955-128</td><td>Methacrylate is required since some procedures are conducted at 340 nm or below</td></tr><tr><td>&amp;beta;-Nicotinamide adenine dinucleotide phosphate sodium salt</td><td>MilliporeSigma</td><td>N0505</td><td></td></tr><tr><td>&amp;beta;-Nicotinamide adenine dinucleotide, reduced disodium salt</td><td>MilliporeSigma</td><td>43420</td><td></td></tr><tr><td>Nucleoside 5&#039;-diphosphate kinase</td><td>MilliporeSigma</td><td>N0379</td><td></td></tr><tr><td>Phosphoenolpyruvate, monopotassium salt</td><td>MilliporeSigma</td><td>P7127</td><td></td></tr><tr><td>Phosphoglucomutase from rabbit muscle</td><td>MilliporeSigma</td><td>P3397</td><td></td></tr><tr><td>Phosphorylase A from rabbit muscle</td><td>MilliporeSigma</td><td>P1261</td><td></td></tr><tr><td>Pyruvate Kinase/Lactic Dehydrogenase enzymes from rabbit muscle</td><td>MilliporeSigma</td><td>P0294</td><td></td></tr><tr><td>UDP-glucose, disodium salt</td><td>MilliporeSigma</td><td>U4625</td><td></td></tr></tbody></table>

spectrophotometric methods for the study of eukaryotic glycogen metabolism

Glycogen is synthesized as a storage form of glucose by a wide array of organisms, ranging from bacteria to animals. The molecule comprises linear chains of &#945;1,4-linked glucose residues with branches introduced through the addition of &#945;1,6-linkages. Understanding how the synthesis and degradation of glycogen are regulated and how glycogen attains its characteristic branched structure requires the study of the enzymes of glycogen storage. However, the methods most commonly used to study these enzyme activities typically employ reagents or techniques that are not available to all investigators. Here, we discuss a battery of procedures that are technically simple, cost-effective, and yet still capable of providing valuable insight into the control of glycogen storage. The techniques require access to a spectrophotometer, operating in the range of 330 to 800 nm, and are described assuming that the users will employ disposable, plastic cuvettes. However, the procedures are readily scalable and can be modified for use in a microplate reader, allowing highly parallel analysis.

Determination of glycogen synthase activity 
Figure 1 shows representative results from glycogen synthase assays using purified enzymes. In panel A, following a slight lag, there was a linear decrease in the absorption at 340 nm over time for a period of around 12 min. The rate of change in absorption in Figure 1A was ~0.12 absorbance units/min. A rate of change in absorbance between ~0.010 and ~0.20 absorbance units/min is optimal and the amount of glycogen synthase added should be adjusted to yield rates within this range. In panel B, the result of adding too much glycogen synthase to the assay is shown. Here, the reaction was complete within the first 2 min. The Control reaction, which in these cases contained no glycogen synthase, showed no measurable decrease in absorbance over time. As elaborated upon in the Discussion, the use of tissue homogenates in this assay is perfectly feasible, although additional Control reactions are required.
The protocol described here uses oyster glycogen as a substrate, which works well with glycogen synthases from many different species. However, it should be noted that glycogen synthases may display quite variable activity depending upon the type of glycogen employed. Therefore, it is advisable to survey a variety of forms of glycogen prior to beginning any detailed study.
The protocol given includes glucose-6-phoposphate in the reaction mixture, since many glycogen synthases are allosterically activated by this compound9,23,24,25,26. Conducting assays in the presence and absence of glucose-6-phosphate (making up the reaction volume with water), allows calculation of the -/+ glucose-6-phosphate activity ratio, which is a useful indication of the phosphorylation state of mammalian and fungal glycogen synthases1,27.
The determination of glycogen synthase activity from the change in absorbance is rather straightforward. The extinction coefficient of NADH is taken as 6220 M-1 cm-1, allowing calculation of the rate of change in NADH concentration from the rate of absorbance change as follows:
A rate of change in absorption of 0.12 units/min corresponds to 0.12/6220 = 1.93 x 10-5 mol/L/min change in NADH concentration. The volume in the cuvette was 0.8 mL, meaning that the change in amount of NADH was: 1.93 x 10-5 x 0.8 x 10-3 = 3.46 x 10-8 mol/min. The volume of enzyme added was 60 &#181;L, 3.46 x 10-8 x (1000 / 60) = 5.76 x 10-7 mol NADH consumed/min/mL enzyme.
Since there is a one-to-one relationship between the NADH consumed and the glucose incorporated into glycogen, the rate of reaction can be expressed as 5.76 x 10-7 mol glucose incorporated/min/mL.
When the protein content of the enzyme sample is known, the specific enzyme activity can be expressed as &#181;mol glucose incorporate/min/mg protein or nmol glucose incorporated/min/mg protein, as appropriate.
As mentioned in the Introduction, the protocol is easily adapted to measure the activity of glycogen synthases that use ADP-glucose as a glucose donor. This is achieved by the simple substitution of UDP-glucose with ADP-glucose in the reaction mixture. Furthermore, both NDP kinase and ATP are omitted from the reaction mixture, since the ADP that is released during glycogen synthase action is a direct substrate for pyruvate kinase.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63046/63046fig01.jpg" /> 
Figure 1: Representative results from assays of glycogen synthase activity. The spectrophotometer was set to take one reading per min for a total time of 20 min. Panel A shows the expected short lag phase followed by a linear decrease in absorbance with time (Experimental). There was no decrease in the absorption noted in the Control reaction. Reaction rate is calculated from the slope of the absorbance change in the linear phase (from 5 to 16 min). Panel B shows the result of adding too much enzyme. Here, the NADH is exhausted within 2 min. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63046/63046fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Determination of glycogen phosphorylase activity 
Figure 2&#160;shows representative data from a glycogen phosphorylase assay using purified enzyme. With the preparation used here, the assay was linear for approximately 3 min. The inset shows a regression line drawn through the points from time 0 to 2.5 min. The slope of this line shows the rate of absorbance change to be 0.022 absorbance units/min. A rate of absorbance increase of around 0.01 to 0.04 is optimal, since the assay will deviate from linearity quite rapidly if too much enzyme is present. The rate of NADPH formation is calculated from the extinction coefficient which, like that of NADH, is 6220 M-1 cm-1. For every 1 mol of NADPH formed, one mol of glucose-1-phosphate had been produced by the action of glycogen phosphorylase. Enzyme activity can therefore be expressed as the amount of glucose-1-phosphate released from glycogen per unit time, following a calculation similar to that outlined above.
The reaction conditions are readily adaptable for those phosphorylases that are sensitive to allosteric modulation. The requisite effectors are simply included in the reaction master mix, replacing some of the water. An important caveat is that the effector itself must be shown not to influence the activity of the coupling enzymes, phosphoglucomutase, and glucose-6-phosphate dehydrogenase.
Lastly, as with glycogen synthase described above, the type of glycogen used as the substrate may impact the rate of the reaction. While oyster glycogen works well with phosphorylases from many species, it may not always be the optimal choice.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63046/63046fig02.jpg" /> 
Figure 2: Representative results from assays of glycogen phosphorylase activity. The spectrophotometer was set to take one reading every 30 s for a total time of 10 min. There was a steady increase in absorbance recorded in the presence of glycogen phosphorylase (Experimental), while the reaction without added phosphorylase remained at baseline (Control). The inset shows an enlargement of the initial reaction period, demonstrating the linearity of product formation with respect to time. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63046/63046fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Determination of glycogen debranching enzyme activity 
The data shown in Figure 3 are representative of a glycogen debranching enzyme assay using purified debranching enzyme. At each time point, the change in absorption that occurred in the absence of added branching enzyme (Control) was subtracted from the change in absorption that occurred in the presence of branching enzyme (Reaction). The resulting absorbance values were then plotted. As above, a rate of change of NADPH concentration is calculated from the initial slope of the curve by regression analysis. In this example, the increase in NADPH per unit time was linear for 10 min, with a slope of 0.0079 absorbance units/min. While these data are perfectly useable, the addition of slightly less enzyme would have given a shallower slope and allowed for a longer linear phase. Alternatively, additional readings could be taken by removing aliquots for measurement at 2 min and 7 min incubation. Determination of debranching enzyme activity is very straightforward, since 1 mol of NADPH is formed for every 1 mol of glucose released by the &#945;1,6-glucosidase activity of debranching enzyme. Thus, reaction rate can be expressed as amount of glucose released from phosphorylase limit dextrin per unit time, following the same type of calculation as was used for the glycogen synthase and phosphorylases assays, above.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63046/63046fig03.jpg" /> 
Figure 3: Representative results from assays of glycogen debranching enzyme activity. Samples of phosphorylase limit dextrin were treated with debranching enzyme for 5, 10, 20, or 40 min. The increase in absorbance at 340 nm, produced as NADP was reduced to NADPH in a coupled enzyme assay, was measured in samples taken at each of these time points. The reaction showed a linear phase, persisting for at least 10 min. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63046/63046fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Determination of glycogen branching enzyme activity 
Figure 4&#160;shows data from glycogen branching enzyme assays. At each of the indicated time points, the absorbance of the Control and Reaction samples were measured. The absorbance of the Reaction sample at 660 nm was subtracted from that of the corresponding Control sample, and the absorbance difference was plotted against time. A regression line was then drawn through the points (panel A). Reaction rate can be expressed simply as the change in absorbance at 660 nm per unit time. The maximum change in absorbance that can occur in this assay is only ~0.4 absorbance units, representing maximal branching of the added amylose by the branching enzyme (panel B). Furthermore, when the absorbance of the Reaction tubes drops more than ~0.2 absorbance units below that of the Control, the assay is no longer within the linear range and no estimation of reaction rate can be made (panel B).
The amylose used as a substrate in this procedure will begin to leave solution quite readily if chilled or frozen and then thawed. Therefore, it is important to make the amylose substrate solution fresh and to ensure that no precipitate has formed prior to use.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63046/63046fig04.jpg" /> 
Figure 4: Representative results from assays of glycogen branching enzyme activity. Samples of amylose were treated with branching enzyme. Aliquots were removed at the time points shown and added to an acidified iodine reagent. The absorbance of amylose/iodine complex formed was then measured at 660 nm. The data shown represent the difference in absorbance between control incubations that lacked branching enzyme and reactions that contained branching enzyme. Panel A shows a decrease in absorbance at 660 nm due to debranching enzyme activity, which was linear for ~20 min. Panel B illustrates the narrow dynamic range of the assay, where the maximum change in absorbance that can be produced is ~0.4 absorbance units and linearity is lost when the change in absorption is ~0.2 absorbance units. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63046/63046fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Qualitative assessment of the extent of glycogen branching 
Amylose, amylopectin, phosphorylase limit dextrin, glycogen isolated from yeast were combined with iodine/saturated calcium chloride solution and the absorption spectra of the resulting complexes were collected (Figure 5). Using the masses of glycogen, amylopectin, and amylose given in the protocol described above, the maximum absorbance reading obtained should be around 0.7 to 0.8, as shown here (panels A and B). The absorbance maxima for amylose and amylopectin are around 660 nm and 500 nm, and 385 nm respectively. Phosphorylase limit dextrin was included here, since collecting the absorbance spectrum of phosphorylase limit dextrin/iodine complexes provides a quick check of the extent of phosphorylase digestion achieved during the preparation of this debranching enzyme substrate. Glycogen from most sources produces two peaks, one at approximately 400 nm and a second peak at 460 nm (Figure 5B). Leftward shifts in the absorbance spectra of glycogen indicate increased branching/decreased outer chain length. Conversely, rightward shifts indicate decreased branching/increased outer chain length.
The saturated calcium chloride solution is dense and the added glycogen samples will form a layer across the top when added. Therefore, careful mixing is needed to obtain a homogenous solution. In addition, if the carbohydrate samples used are not fully dissolved before mixing with the calcium chloride solution, dark-staining aggregates will form in the cuvette. These aggregates will obviously impede collection of an absorption spectrum and it is important to ensure that the solution in the cuvette is clear before proceeding with any measurements.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63046/63046fig05.jpg" /> 
Figure 5: Representative results from the qualitative assessment of glycogen branching. Samples of purified phosphorylase limit dextrin, amylopectin, amylose (Panel A) or glycogen (Panel B) were combined with iodine/saturated calcium chloride solution and the absorption spectra of the resulting complexes were measured from 330 nm to 800 nm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63046/63046fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism at JoVE.com

Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism

Techniques to measure the activity of key enzymes of glycogen metabolism are presented, using a simple spectrophotometer operating in the visible range.

Glycogen is synthesized as a storage form of glucose by a wide array of organisms, ranging from bacteria to animals. The molecule comprises linear chains ...

spectrophotometric-methods-for-study-eukaryotic-glycogen

Nanyang Technological University

Education

Research

JoVE Core

JoVE Journal

Biochemistry

Anatomy and Physiology

Analytical Chemistry

Unit 1

Chemical Applications of Statistical Analyses

Absorption of Nutrients

Biochemistry of the Cell

SCIENTISTS IN ACTION

3.6K Views.  Des Moines University. De protocollen zijn belangrijk, omdat ze het gebruik van radioactieve isotopen vermijden en een barrière verwijderen die veel werknemers kan beletten enzymen van glycogeenmetabolisme te bestuderen. De beschreven procedures zijn goedkoop en vereisen alleen toegang tot een eenvoudige spectrofotometer. Om te beginnen met de bepaling van glycogeensynthase-activiteit, ontdooi de eerder bereide stamoplossingen van UDP-glucose, ATP, fosfoenolpyruvaat en NDP-kinase op ijs.Verwarm een waterbad...

Video: Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism

Determination of the Glycogen Content in Cyanobacteria

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have highlighted complex mechanisms of glycogen metabolism, including the diel cycle of biosynthesis and catabolism, redox regulation, and the involvement of non-coding RNA. At the same time, efforts are being made to redirect carbon from glycogen to desirable products in genetically engineered cyanobacteria to enhance product yields. Several methods are used to determine the glycogen contents in cyanobacteria, with variable accuracies and technical complexities. Here, we provide a detailed protocol for the reliable determination of the glycogen content in cyanobacteria that can be performed in a standard life science laboratory. The protocol entails the selective precipitation of glycogen from the cell lysate and the enzymatic depolymerization of glycogen to generate glucose monomers, which are detected by a glucose oxidase-peroxidase (GOD-POD) enzyme coupled assay. The method has been applied to Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002, two model cyanobacterial species that are widely used in metabolic engineering. Moreover, the method successfully showed differences in the glycogen contents between the wildtype and mutants defective in regulatory elements or glycogen biosynthetic genes.

Here, we present a reliable and easy assay to measure the glycogen content in cyanobacterial cells. The procedure entails precipitation, selectable depolymerization, and the detection of glucose residues. This method is suitable for both wildtype and genetically engineered strains and can facilitate the metabolic engineering of cyanobacteria.

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Immunology and Infection

The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are influenced by its structure. Hence, a suitable extraction method that isolates representative samples of glycogen is crucial to the study of this macromolecule. Compared to other extraction methods, a method that employs a sucrose density gradient centrifugation step can minimize molecular damage. Based on this method, a recent publication describes how the density of the sucrose solution used during centrifugation was varied (30%, 50%, 72.5%) to find the most suitable concentration to extract glycogen particles of a wide variety of sizes, limiting the loss of smaller particles. A 10 min boiling step was introduced to test its ability to denature glycogen degrading enzymes, thus preserving glycogen. The lowest sucrose concentration (30%) and the addition of the boiling step were shown to extract the most representative samples of glycogen.

An optimal sucrose concentration was determined for the extraction of liver glycogen using sucrose density gradient centrifugation. The addition of a 10 min boiling step to inhibit glycogen-degrading enzymes proved beneficial.

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are ...

Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This enables quantifications of ultrastructural aspects such as volume fractions, surface area to volume ratios, morphometry, and physical contact sites of different subcellular structures. In the 1970s, a protocol for enhanced staining of glycogen in cells was developed and paved the way for a string of studies on the subcellular localization of glycogen and glycogen particle size using transmission electron microscopy. While most analyses interpret glycogen as if it is homogeneously distributed within the muscle fibers, providing only a single value (e.g., an average concentration), transmission electron microscopy has revealed that glycogen is stored as discrete glycogen particles located in distinct subcellular compartments. Here, the step-by-step protocol from tissue collection to the quantitative determination of the volume fraction and particle diameter of glycogen in the distinct subcellular compartments of individual skeletal muscle fibers is described. Considerations on how to 1) collect and stain tissue specimens, 2) perform image analyses and data handling, 3) evaluate the precision of estimates, 4) discriminate between muscle fiber types, and 5) methodological pitfalls and limitations are included.

A modified post-fixation procedure increases the contrast of glycogen particles in tissue. This paper provides a step-by-step protocol describing how to handle the tissue, conduct the imaging, and use stereological methods to obtain unbiased and quantitative data on fiber type-specific subcellular glycogen distribution in skeletal muscle.

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This ...

Determination of Glucan Chain Length Distribution of Glycogen Using the Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) Method

Glycogen particles are branched polysaccharides composed of linear chains of glucosyl units linked by &#945;-1,4 glucoside bonds. The latter are attached to each other by &#945;-1,6 glucoside linkages, referred to as branch points. Among the different forms of carbon storage (i.e., starch, &#946;-glucan), glycogen is probably one of the oldest and most successful storage polysaccharides found across the living world. Glucan chains are organized so that a large amount of glucose can quickly be stored or fueled in a cell when needed. Numerous complementary techniques have been developed over the last decades to solve the fine structure of glycogen particles. This article describes Fluorophore-Assisted Carbohydrate Electrophoresis (FACE). This method quantifies the population of glucan chains that compose a glycogen particle. Also known as chain length distribution (CLD), this parameter mirrors the particle size and the percentage of branching. It is also an essential requirement for the mathematical modeling of glycogen biosynthesis.

Determination of Glucan Chain Length Distribution of Glycogen Using the Fluorophore-Assisted Carbohydrate Electrophoresis FACE Method

In the present protocol, the Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) technique is used to determine the chain length distribution (CLD) and the average chain length (ACL) of glycogen.

Glycogen particles are branched polysaccharides composed of linear chains of glucosyl units linked by &#945;-1,4 glucoside bonds. The latter are attached ...

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.

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.

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 ...

Biology

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

A Non-Degradative Extraction Method for Molecular Structure Characterization of Bacterial Glycogen Particles

Currently, there exist a variety of glycogen extraction methods, which either damage glycogen spatial structure or only partially extract glycogen, leading to the biased characterization of glycogen fine molecular structure. To understand the dynamic changes of glycogen structures and the versatile functions of glycogen particles in bacteria, it is essential to isolate glycogen with minimal degradation. In this study, a mild glycogen isolation method is demonstrated by using cold-water (CW) precipitation via sugar density gradient ultra-centrifugation (SDGU-CW). The traditional trichloroacetic acid (TCA) method and potassium hydroxide (KOH) method were also performed for comparison. A commonly used lab strain, Escherichia coli BL21(DE3), was used as a model organism in this study for demonstration purposes. After extracting glycogen particles using different methods, their structures were analyzed and compared through size exclusion chromatography (SEC) for particle size distribution and fluorophore-assisted capillary electrophoresis (FACE) for linear chain length distributions. The analysis confirmed that glycogen extracted via SDGU-CW had minimal degradation.

Bacterial glycogen structure is greatly impacted by extraction methods which may result in molecular degradation and/or biased sampling. It is essential to develop methods to minimize these problems. Here, four extraction methods have been compared using size distribution and chain length distribution as key criteria for minimizing extraction artifacts.

Currently, there exist a variety of glycogen extraction methods, which either damage glycogen spatial structure or only partially extract glycogen, ...

Transmission Electron Microscopy to Quantify Glycogen Distribution in Human Skeletal Muscles

Source: Jensen, R., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63347">Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy.</a> J. Vis. Exp. (2022)
In this video, we demonstrate the sample preparation of human skeletal muscle tissue and its staining for transmission electron microscopy to visualize and quantify subcellular glycogen distribution.

Source: Jensen, R., et al. Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy. J. Vis. ...

JoVE Encyclopedia of Experiments

Biological Techniques

Microscopy Techniques

Spectrophotometric Assay to Determine Glycogen Synthase Activity

Source:&#160; Wilson W.A., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63046">Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism.</a> J. Vis. Exp. (2021).
This video describes a spectrophotometric assay for the estimation of glycogen synthase activity. The method relies on the reduction in the absorbance of NADH with time and indicates glycogen synthase activity.

Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism

Samenvatting

Meer video's verkennen