Name: The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination (Video) | JoVE Journal
Uploaded: 2022-02-08T14:30:00.000+00:00
Duration: 4 min 50 s
Description: Learn to extract liver glycogen while preserving its structure and minimizing loss of small particles using this optimized boiling protocol. This method enables research on diabetes and glycogen storage diseases.

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5 minutes

The authors are grateful to Mr. Gaosheng Wu and Miss Yunwen Zhu for technical assistance with FACE and Mr. Zhenxia Hu and Mr. Dengbin for technical assistance with SEC. MAS is supported by an Advance Queensland Industry Research Fellowship, Mater Foundation, Equity Trustees, and the L G McCallam Est and George Weaber Trusts. This work was supported by the Priority Academic Program of Jiangsu Higher Education Institutions, a Natural Science Foundation of China grant C1304013151101138, and the 2017 Jiangsu Innovation and Entrepreneurship talents program. Figure 1-5 were created using BioRender.

Mouse livers used to optimize this procedure21 were from 12 male BKS-DB/Nju background mice (7.2 weeks old, see the Table of Materials). Animal use was approved by Renmin Hospital of Wuhan University Animal Care and Ethics Committee, IACUC Issue No. WDRM 20181113.
1 Animal tissues
<ol>
	<li>Weigh mouse liver (1-1.8 g of whole liver from each mouse).</li>
	<li>Rapidly freeze the mouse liver in liquid nitrogen and store it at -80 &#176;C.</li>
</ol>
2. Preparation of buffer and reagents
<ol>
	<li>Prepare glycogen isolation buffer containing 5 mM Tris,150 mM NaCl, 2 mM EDTA, 50 mM NaF, and 5 mM sodium pyrophosphate with deionized water, and adjust the pH to 8.</li>
	<li>Prepare 30% (w/w) sucrose solution (found to be most optimal for liver glycogen21).</li>
	<li>Prepare sodium acetate buffer (1 M, pH 4.5), acetic acid buffer (0.1 M, pH 3.5), sodium hydroxide solution (0.1 M), and sodium cyanoborohydride (1 M).</li>
	<li>Prepare 8-aminopyrene-1,3,6-trisulfonate (APTS) solution by adding 5 mg of APTS to 50 &#181;L of 15% glacial acetic acid.</li>
	<li>Prepare ammonium nitrate solution containing 50 mM ammonium nitrate with 0.02% sodium azide.</li>
</ol>
3. Glycogen extraction (Figure 2)
<ol>
	<li>Transfer the frozen liver tissue (~1 g) to a 15 mL tube containing 6 mL of glycogen isolation buffer.</li>
	<li>Keeping it on ice, homogenize the liver tissue using a tissue homogenizer.</li>
	<li>Transfer half of the suspension (3 mL) to a new tube and boil for 10 min (shown to be optimal for glycogen structural studies21). Keep the other half of the suspension (3 mL) on ice to extract glycogen containing associated proteins that are not denatured. 
	NOTE: Unboiled samples should always be kept on ice during glycogen extraction steps. If glycogen proteins are not important for study, the whole sample can undergo the 10 min boiling step.</li>
	<li>Remove an 8 &#181;L aliquot from each tube, keep the aliquots on ice, and use them for the glycogen content determination (see section 4).</li>
	<li>Centrifuge the remaining suspension at 6,000 &#215; g for 10 min at 4 &#176;C.</li>
	<li>Transfer the supernatants to ultracentrifuge tubes, and centrifuge them at 3.6 &#215; 105&#160;g for 90 min at 4 &#176;C.</li>
	<li>Discard the remaining supernatants and resuspend the pellets in 1.5 mL of glycogen isolation buffer.</li>
	<li>Layer the samples over 1.5 mL of 30% sucrose solution in 4 mL ultracentrifuge tubes and centrifuge at 3.6 &#215; 105&#160;g for 2 h at 4 &#176;C.</li>
	<li>Discard the remaining supernatants and resuspend the pellets in 200 &#181;L of deionized water.</li>
	<li>Add 800 &#181;L of absolute ethanol to the suspensions and mix well to precipitate glycogen23,24. Store the mixtures at -20 &#176;C for at least 1 h to allow precipitation.</li>
	<li>Centrifuge the samples at 6,000 &#215; g for 10 min at 4 &#176;C. Discard the supernatants and resuspend the pellets in 200 &#181;L of deionized water.</li>
	<li>Repeat this ethanol precipitation process 3x and resuspend the final glycogen pellet in 200 &#181;L of deionized water.</li>
	<li>Remove an aliquot of 8 &#181;L from each tube for glycogen content determination (see section 4).</li>
	<li>Freeze the remaining supernatants in liquid nitrogen and freeze-dry (lyophilize) overnight. Store the dry glycogen samples in the freezer at -20 &#176;C. 
	&#8203;NOTE: The dry glycogen samples should be stable at -20 &#176;C; however, there are no data to indicate how long they last without any structural changes.</li>
</ol>
4. Glycogen content determination (Figure 3)
<ol>
	<li>Add 8 &#181;L of the glycogen supernatants, (see sections 3.13 and 3.4), 5 &#181;L of amyloglucosidase (3269 U/mL), and 100 &#181;L of sodium acetate buffer (1 M, pH 4.5) to a microcentrifuge tube and fill the tube to the 500 &#181;L mark with deionized water.</li>
	<li>Prepare controls that use deionized water instead of amyloglucosidase.</li>
	<li>Incubate the samples at 50 &#176;C for 30 min, while keeping the controls on ice.</li>
	<li>Centrifuge at 6,000 &#215; g at 4 &#176;C for 10 min, and mix 300 &#181;L of each resulting supernatant with 1 mL of glucosidase oxidase/peroxidase (GOPOD) reagent.</li>
	<li>Construct a calibration curve by mixing 300 &#181;L of denionized water containing 0, 10, 20, 30, 40, and 50 &#181;g of D-glucose with 1 mL of GOPOD reagent.</li>
	<li>Incubate the mixtures at 50 &#176;C for a further 30 min.</li>
	<li>Read the absorbance (510 nm) of each sample using 96-well plates (150 &#181;L per well) using a UV-vis spectrophotometer.</li>
	<li>Subtract the absorbances of control samples (with no amyloglucosidase) from the absorbances of experimental samples, then calculate the glycogen content based on the D-glucose standard curve.</li>
</ol>
5. Crude yield, glycogen yield, and purity determination
<ol>
	<li>For the crude yield, weigh the freeze-dried glycogen sample and calculate the yield as a percentage of the wet liver tissue. 
	NOTE: This yield should be adjusted to correct for the aliquots taken in each glycogen content step.</li>
	<li>For glycogen purity, determine the glycogen content in the final pellets, as described in section 4. Calculate the purity as a percentage of the determined glycogen content relative to the crude yield (see step 5.1).</li>
	<li>For glycogen yield, determine the glycogen content of the homogenized samples without boiling and before any centrifugation, as described in section 4. Calculate the glycogen yield as a percentage of the glycogen content in the final pellets (see step 5.2) to that of the glycogen content determined in the initial homogenate.</li>
</ol>
6. Analysis of chain-length distributions (Figure 4)
<ol>
	<li>Weigh 0.5 mg of freeze-dried glycogen in 1.5 mL tubes.</li>
	<li>Add 90 &#181;L of deionized water and 1.5 &#181;L of sodium azide (0.04 g/mL) to the tubes.</li>
	<li>Add 5 &#181;L of acetic acid buffer (0.1 M, pH 3.5) and 2 &#181;L of isoamylase solution (180 U/mg) to the tubes to debranch the glycogen.</li>
	<li>Incubate the samples at 37 &#176;C for 3 h.</li>
	<li>Add 5 &#181;L of sodium hydroxide solution (0.1 M) to the samples to increase the pH to 7.0.</li>
	<li>Freeze the samples in liquid nitrogen and freeze-dry (lyophilize) overnight.</li>
	<li>Add 2 &#181;L of APTS solution (5 mg of APTS in 50 &#181;L of 15% glacial acetic acid) and 2 &#181;L sodium cyanoborohydride (1 M) to the freeze-dried debranched glycogen.</li>
	<li>Centrifuge the samples at 4,000 &#215; g for 2 min.</li>
	<li>Incubate the samples at 60 &#176;C for 3 h in the dark. 
	NOTE: The tube can be covered with aluminum foil to protect the contents from light.</li>
	<li>Add 200 &#181;L of deionized water to the samples and vortex them until all precipitate is dissolved.</li>
	<li>Centrifuge the samples at 4,000 &#215; g for 2 min.</li>
	<li>Transfer aliquots of 50 &#181;L to fluorophore-assisted carbohydrate electrophoresis (FACE) micro-vials for analysis. 
	&#8203;NOTE: The data are shown as the relative abundance of (debranched) chains (Nde(X)) for each degree of polymerization (DP, symbol X).</li>
</ol>
7. Analysis of molecular size distributions (Figure 5)
<ol>
	<li>Dissolve 0.5 mg of freeze-dried glycogen in 50 mM ammonium nitrate and 0.02% sodium azide at 1 mg/mL.</li>
	<li>Incubate the samples in a thermomixer at 80 &#176;C for 3 h at 300 rpm.</li>
	<li>Inject the samples into an SEC system using a pre-column and 1000 &#197; and 10,000 &#197; columns at 80 &#176;C with a flow rate of 0.3 mL/min (see the Table of Materials). Use a refractive index detector to determine the relative weight of molecules at each elution volume.</li>
	<li>Using pullulan standards (PSS) with molar masses ranging from 342 to 1.22 &#215; 106,&#160;plot an SEC universal calibration curve to convert the elution time to Rh (hydrodynamic radius). Express the data from the differential refractive index (DRI) detector as an SEC weight distribution w (log Rh) as a function of Rh.</li>
</ol>

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<li>Parker, G. J., Koay, A., Gilbert-Wilson, R., Waddington, L. J., Stapleton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((AMP-activated+protein+kinase+does+not+associate+with+glycogen+alpha-particles+from+rat+liver%5BTitle%5D)+AND+%22Biochemical+and+Biophysical+Research+Communications%22%5BJournal%5D)">AMP-activated protein kinase does not associate with glycogen alpha-particles from rat liver.</a> Biochemical and Biophysical Research Communications. 362, 811-815 (2007).</li>
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</ol>

The authors have no conflicts of interest to disclose.

Previous studies have shown that the structure of glycogen is important for its properties; for example, the molecular size affects the degradation rate of glycogen10, and the chain length distribution affects its solubility26. To properly understand these relationships, it is important to extract glycogen with a procedure that isolates, as much as possible, a representative and undamaged sample. Traditional methods of extraction utilized either hot alkaline conditions or c...

Glycogen is a complex, hyperbranched polymer of glucose found in animals, fungi, and bacteria1. In mammals, liver glycogen functions as a blood glucose buffer, preserving homeostasis, while muscle glycogen acts as a short-term glucose reservoir to provide energy directly2. The structure of glycogen is often described by three levels (shown in Figure 1): 1. Linear chains are formed by glucose monomers via (1&#8594;4)-&#945; glycosidic bonds, with branch points being connected via (1&#8594;6)-&#945; glycosidic bonds; 2. highly branched &#946; particles (~20 nm in diameter) that, especially in tissues such as skeletal muscle, act as independent glycogen molecules3,4; 3. larger &#945; glycogen particles (up to 300 nm in diameter) that consist of smaller &#946; glycogen units, which are found in the liver5, heart6, and in some non-mammalian species7. Hepatic &#945; particles from diabetic mice are molecularly fragile, with a propensity to degrade to &#946;-particles when dissolved in dimethyl sulfoxide (DMSO), while &#945; particles from non-diabetic controls generally remain unchanged. One hypothesis is that this fragility may exacerbate the poor blood glucose balance seen in diabetes, with the fragile &#945; particles potentially resulting in higher proportions of the more rapidly degraded &#946; particle8,9,10,11.
Traditional glycogen extraction methods utilize the relatively harsh conditions of exposing the liver tissue to hot alkaline solution12 or acid solutions such as trichloroacetic acid (TCA)13 or perchloric acid (PCA)14. While effective at separating the glycogen from other components of the liver tissue, these methods inevitably degrade the glycogen structure to some extent15,16. Although these methods are suitable for quantitative measurement of the glycogen content, they are not ideal for studies focused on obtaining structural information on the glycogen due to this structural damage. Since the development of these methods, a milder extraction procedure has been developed that utilizes cold Tris buffer (shown to inhibit glucosidase degradation) with sucrose density gradient ultracentrifugation17,18,19. With the pH controlled at ~8, this method does not subject the glycogen to the acid or alkaline hydrolysis seen in previous procedures.
Sucrose density gradient ultracentrifugation of homogenized liver tissue can separate glycogen particles from the majority of cell material. If necessary, additional purification can be performed by preparative size exclusion chromatography, resulting in the collection of purified glycogen with attached glycogen-associating proteins20. Although this method, with milder conditions, is more likely to preserve the structure of glycogen, it is difficult to prevent some portion of the glycogen from being lost in the supernatant, especially smaller glycogen particles that are less dense15. Another potential cause of glycogen loss is that the milder conditions allow some enzymatic degradation, resulting in lower glycogen yields compared to harsher extraction methods. Recent research reported optimization of the liver-glycogen extraction method to preserve the structure of glycogen21. Here, various sucrose concentrations (30%,50%, 72.5%) were tested to determine whether lower sucrose concentrations minimized the loss of smaller glycogen particles. The rationale was that the lower density would allow for smaller, less dense particles to penetrate the sucrose layer and aggregate in the pellet with the rest of the glycogen.
In this study, the extraction methods with and without a 10 min boiling step were compared to test whether glycogen degradation enzymes could be denatured, resulting in the extraction of more glycogen that was also free from partial degradation. Whole molecular size distributions and the glycogen chain length distributions were used to determine the structure of the extracted glycogen, similar to a starch extraction optimization published previously22. Size exclusion chromatography (SEC) with differential refractive index (DRI) detection was used to obtain the size distributions of glycogen, which describe the total molecular weight as a function of molecular size. Fluorophore-assisted carbohydrate electrophoresis (FACE) was used to analyze the chain-length distributions, which describe the relative number of glucoside chains of each given size (or degree of polymerization). This paper describes the methodology of extracting glycogen from liver tissues based on the previous optimization study21. The data suggest that the method most suited to preserve glycogen structure is a sucrose concentration of 30% with a 10 min boiling step.

<table><tbody><tr><td>8-aminopyrene-1,3,6-trisulfonate (APTS)</td><td>SIGMA Aldrich</td><td>9341</td><td>0.1 M solution</td></tr><tr><td>Acetic acid</td><td>SIGMA Aldrich</td><td>695092</td><td>0.1 M, pH 3.5 solution</td></tr><tr><td>Agilent 1260 Infinity SEC system</td><td>Agilent, Santa Clara, CA, USA</td><td></td><td>Size-exclusion chromatography (SEC)</td></tr><tr><td>BKS-DB/Nju background mice</td><td>Nanjing Biomedical Research Institution of Nanjing University</td><td></td><td></td></tr><tr><td>D-Glucose Assay Kit (GOPOD Format)</td><td>Megazyme</td><td>K-GLUC</td><td></td></tr><tr><td>Ethylenedinitrilotetraacetic acid (EDTA)</td><td>SIGMA Aldrich</td><td>431788</td><td></td></tr><tr><td>Homogenizer</td><td>IKA</td><td>T 25</td><td></td></tr><tr><td>Hydrochloric acid</td><td>SIGMA Aldrich</td><td>2104</td><td>0.1 M solution</td></tr><tr><td>Hydrochloric acid</td><td>SIGMA Aldrich</td><td>2104</td><td>0.1 M solution</td></tr><tr><td>P/ACE MDQ plus system</td><td>Ab Sciex, US</td><td></td><td>Fluorophore-assisted carbohydrate electrophoresis (FACE)</td></tr><tr><td>Refractive index detector</td><td>Optilab UT-rEX, Wyatt, Santa Barbara, CA, USA)</td><td></td><td>Size-exclusion chromatography (SEC)</td></tr><tr><td>Sodium acetate</td><td>SIGMA Aldrich</td><td>241245</td><td>1 M, pH 4.5 solution</td></tr><tr><td>Sodium azide</td><td>SIGMA Aldrich</td><td>S2002</td><td></td></tr><tr><td>Sodium chloride</td><td>SIGMA Aldrich</td><td>S9888</td><td></td></tr><tr><td>Sodium cyanoborohydride</td><td>SIGMA Aldrich</td><td>156159</td><td>1 M solution</td></tr><tr><td>Sodium fluoride</td><td>SIGMA Aldrich</td><td>201154</td><td></td></tr><tr><td>Sodium hydroxide</td><td>SIGMA Aldrich</td><td>43617</td><td>0.1 M solution</td></tr><tr><td>Sodium nitrate</td><td>SIGMA Aldrich</td><td>NISTRM8569</td><td></td></tr><tr><td>Sodium pyrophosphate</td><td>SIGMA Aldrich</td><td>221368</td><td></td></tr><tr><td>Sucrose</td><td>SIGMA Aldrich</td><td>V90016</td><td></td></tr><tr><td>SUPREMA pre-column, 1,000 and 10,000 columns</td><td>Polymer Standards Services, Mainz, Germany</td><td></td><td>Size-exclusion chromatography (SEC)</td></tr><tr><td>Trizma</td><td>SIGMA Aldrich</td><td>T 1503</td><td></td></tr><tr><td>Ultracentrifuge tubes</td><td>Beckman</td><td></td><td>4 mL, Open-Top Thinwall Ultra-Clear Tube, 11 x 60 mm</td></tr></tbody></table>

the extraction of liver glycogen molecules for glycogen structure determination

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.

While the procedure described above is for the most optimal method (30% sucrose with the addition of a 10min boiling step), data are provided here for glycogen extracted via three sucrose concentrations (30%, 50%, 72.5%), with and without a boiling step, as previously described21. Following the protocol, the purity, crude yield, and glycogen yield of dry glycogen extracted by different conditions are given in Table 1, reproduced from21. There were no signif...

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 ...

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Research

JoVE Journal

Biochemistry

In this video, we describe a qualitative spectrophotometric assay to determine the extent of branching in a glycogen sample with an uncharacterized structure. Comparing the absorbance maxima of the uncharacterized sample with that of polysaccharides with known branched structures indicates the presence of branching in the uncharacterized sample.

Glycogen Branching Assay to Measure the Degree of Glycogen Branching

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.

Transmission Electron Microscopy to Quantify Glycogen Distribution in Human Skeletal Muscles

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

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.

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

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.

Determination of the Glycogen Content in Cyanobacteria

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

Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism

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.

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

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.

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

This protocol details the key steps to enable rigorous and reproducible measurement of both glycogen and N-linked glycans using mass spectrometry imaging.

Spatial Molecular Imaging of the Glycome Using Mass Spectrometry

Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein.

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Learn to extract liver glycogen while preserving its structure and minimizing loss of small particles using this optimized boiling protocol. This method enables research on diabetes and glycogen storage diseases.

The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination

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