The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination

Ziyi Wang; Qinghua Liu; Liang Wang; Robert G. Gilbert; Mitchell  A. Sullivan

doi:10.3791/63088

JoVE Journal > Biochemistry

生化学

The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination

Published: February 08, 2022

doi:

10.3791/63088

Ziyi Wang^1,2, Qinghua Liu^3,4, Liang Wang^3,5, Robert G. Gilbert^1,2,6, Mitchell A. Sullivan⁷

¹School of Chemistry and Molecular Biosciences,The University of Queensland, ²Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation,The University of Queensland, ³Jiangsu Provincial Key Laboratory of New Drug Research and Clinical Pharmacy, School of Pharmacy,Xuzhou Medical University, ⁴Department of Pharmaceutical Analysis, School of Pharmacy,Xuzhou Medical University, ⁵Department of Bioinformatics, School of Medical Informatics and Engineering,Xuzhou Medical University, ⁶Joint International Research Laboratory of Agriculture and Agri-Product Safety, College of Agriculture,Yangzhou University, ⁷Glycation and Diabetes Group,Mater Research Institute-The University of Queensland, Translational Research Institute

概要

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.

Abstract

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.

Introduction

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→4)-α glycosidic bonds, with branch points being connected via (1→6)-α glycosidic bonds; 2. highly branched β particles (~20 nm in diameter) that, especially in tissues such as skeletal muscle, act as independent glycogen molecules³^,⁴; 3. larger α glycogen particles (up to 300 nm in diameter) that consist of smaller β glycogen units, which are found in the liver⁵, heart⁶, and in some non-mammalian species⁷. Hepatic α particles from diabetic mice are molecularly fragile, with a propensity to degrade to β-particles when dissolved in dimethyl sulfoxide (DMSO), while α 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 α particles potentially resulting in higher proportions of the more rapidly degraded β 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.

Protocol

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

Weigh mouse liver (1-1.8 g of whole liver from each mouse).
Rapidly freeze the mouse liver in liquid nitrogen and store it at -80 °C.

2. Preparation of buffer and reagents

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.
Prepare 30% (w/w) sucrose solution (found to be most optimal for liver glycogen²¹).
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).
Prepare 8-aminopyrene-1,3,6-trisulfonate (APTS) solution by adding 5 mg of APTS to 50 µL of 15% glacial acetic acid.
Prepare ammonium nitrate solution containing 50 mM ammonium nitrate with 0.02% sodium azide.

3. Glycogen extraction (Figure 2)

Transfer the frozen liver tissue (~1 g) to a 15 mL tube containing 6 mL of glycogen isolation buffer.
Keeping it on ice, homogenize the liver tissue using a tissue homogenizer.
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.
Remove an 8 µL aliquot from each tube, keep the aliquots on ice, and use them for the glycogen content determination (see section 4).
Centrifuge the remaining suspension at 6,000 × g for 10 min at 4 °C.
Transfer the supernatants to ultracentrifuge tubes, and centrifuge them at 3.6 × 10⁵ g for 90 min at 4 °C.
Discard the remaining supernatants and resuspend the pellets in 1.5 mL of glycogen isolation buffer.
Layer the samples over 1.5 mL of 30% sucrose solution in 4 mL ultracentrifuge tubes and centrifuge at 3.6 × 10⁵ g for 2 h at 4 °C.
Discard the remaining supernatants and resuspend the pellets in 200 µL of deionized water.
Add 800 µL of absolute ethanol to the suspensions and mix well to precipitate glycogen²³^,²⁴. Store the mixtures at -20 °C for at least 1 h to allow precipitation.
Centrifuge the samples at 6,000 × g for 10 min at 4 °C. Discard the supernatants and resuspend the pellets in 200 µL of deionized water.
Repeat this ethanol precipitation process 3x and resuspend the final glycogen pellet in 200 µL of deionized water.
Remove an aliquot of 8 µL from each tube for glycogen content determination (see section 4).
Freeze the remaining supernatants in liquid nitrogen and freeze-dry (lyophilize) overnight. Store the dry glycogen samples in the freezer at -20 °C.
NOTE: The dry glycogen samples should be stable at -20 °C; however, there are no data to indicate how long they last without any structural changes.

4. Glycogen content determination (Figure 3)

Add 8 µL of the glycogen supernatants, (see sections 3.13 and 3.4), 5 µL of amyloglucosidase (3269 U/mL), and 100 µL of sodium acetate buffer (1 M, pH 4.5) to a microcentrifuge tube and fill the tube to the 500 µL mark with deionized water.
Prepare controls that use deionized water instead of amyloglucosidase.
Incubate the samples at 50 °C for 30 min, while keeping the controls on ice.
Centrifuge at 6,000 × g at 4 °C for 10 min, and mix 300 µL of each resulting supernatant with 1 mL of glucosidase oxidase/peroxidase (GOPOD) reagent.
Construct a calibration curve by mixing 300 µL of denionized water containing 0, 10, 20, 30, 40, and 50 µg of D-glucose with 1 mL of GOPOD reagent.
Incubate the mixtures at 50 °C for a further 30 min.
Read the absorbance (510 nm) of each sample using 96-well plates (150 µL per well) using a UV-vis spectrophotometer.
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.

5. Crude yield, glycogen yield, and purity determination

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.
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).
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.

6. Analysis of chain-length distributions (Figure 4)

Weigh 0.5 mg of freeze-dried glycogen in 1.5 mL tubes.
Add 90 µL of deionized water and 1.5 µL of sodium azide (0.04 g/mL) to the tubes.
Add 5 µL of acetic acid buffer (0.1 M, pH 3.5) and 2 µL of isoamylase solution (180 U/mg) to the tubes to debranch the glycogen.
Incubate the samples at 37 °C for 3 h.
Add 5 µL of sodium hydroxide solution (0.1 M) to the samples to increase the pH to 7.0.
Freeze the samples in liquid nitrogen and freeze-dry (lyophilize) overnight.
Add 2 µL of APTS solution (5 mg of APTS in 50 µL of 15% glacial acetic acid) and 2 µL sodium cyanoborohydride (1 M) to the freeze-dried debranched glycogen.
Centrifuge the samples at 4,000 × g for 2 min.
Incubate the samples at 60 °C for 3 h in the dark.
NOTE: The tube can be covered with aluminum foil to protect the contents from light.
Add 200 µL of deionized water to the samples and vortex them until all precipitate is dissolved.
Centrifuge the samples at 4,000 × g for 2 min.
Transfer aliquots of 50 µL to fluorophore-assisted carbohydrate electrophoresis (FACE) micro-vials for analysis.
NOTE: The data are shown as the relative abundance of (debranched) chains (N_de(X)) for each degree of polymerization (DP, symbol X).

7. Analysis of molecular size distributions (Figure 5)

Dissolve 0.5 mg of freeze-dried glycogen in 50 mM ammonium nitrate and 0.02% sodium azide at 1 mg/mL.
Incubate the samples in a thermomixer at 80 °C for 3 h at 300 rpm.
Inject the samples into an SEC system using a pre-column and 1000 Å and 10,000 Å columns at 80 °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.
Using pullulan standards (PSS) with molar masses ranging from 342 to 1.22 × 10⁶, plot an SEC universal calibration curve to convert the elution time to R_h(hydrodynamic radius). Express the data from the differential refractive index (DRI) detector as an SEC weight distribution w (log R_h) as a function of R_h.

Representative Results

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 significant differences in crude yield and glycogen yield between the groups extracted with the various conditions. In contrast, the glycogen purity was significantly influenced by both the sucrose concentrations (Table 1, P < 0.001) and by the addition of a boiling step (Table 1, P < 0.0001). Glycogen with the highest purity was extracted using the 30% sucrose concentration along with a 10 min boiling step, which is why it was determined to be the most optimal out of the conditions tested.

Molecular size distributions were used to assess the effects of the various conditions on the size of molecules in the final extract. These were obtained using an aqueous SEC system, as described previously²⁵. Normalizing each distribution to the same maximum value allowed the relative proportion of α to β particles to be compared from each method, shown in Figure 6, reproduced from²¹. The R_h at which the maxima occurs (R_h,max) and the average R_h () are shown in Table 2, reproduced from²¹. Glycogen molecules with R_h < 30 nm were defined as β particles¹¹. The β particle content was calculated as the area under the curve (AUC) of (R_h < 30 nm)/AUC (total R_h). The boiled samples had lower average R_hvalues and a higher β particle content than the unboiled samples (Table 2, P < 0.05). Lower sucrose concentrations resulted in lower values and higher β particle contents (Table 2, P <0.05). The introduction of a boiling step also led to lower R_h,maxvalues (Table 2, P<0.05), while the sucrose concentration had no significant effect.

Chain-length distributions (CLDs) provide the relative number of chains of each given length (number of connected glucose units, or degree of polymerization), obtained using FACE. The CLDs are shown in Figure 7, reproduced using data from²¹. The number-average chain length (ACL) was calculated as (Σ_X X N_de(X))/ (Σ_X N_de(X)) (Table 2). The ACLs of unboiled samples were significantly smaller and more varied than those of the boiled samples (Table 2, P < 0.05). However, the sucrose concentration did not significantly affect the ACLs. This supported the hypothesis that boiling the samples for 10 min as a pre-extraction step would preserve the glycogen structure. The proposed mechanism is the denaturing of glycogen-degrading enzymes.

Figure 1: The three levels of glycogen structure. Please click here to view a larger version of this figure.

Figure 2: Glycogen extraction process. Steps to extract and purify the glycogen from mouse liver. Please click here to view a larger version of this figure.

Figure 3: Glycogen content determination. Steps to determine the glycogen content in liver homogenate, purified dry glycogen or glycogen solution. Please click here to view a larger version of this figure.

Figure 4: Analysis of chain-length distributions. Steps to analyze chain-length distributions by a fluorophore-assisted carbohydrate electrophoresis system. Please click here to view a larger version of this figure.

Figure 5: Analysis of molecular size distributions. Steps to analyze the molecular size distributions by an aqueous size-exclusion chromatography system. Please click here to view a larger version of this figure.

Figure 6: SEC weight distributions of whole (not debranched) mouse liver glycogen. Extraction was performed under different conditions, normalized to have the same maximum in w(log R_h). Each liver homogenate was divided into six equal volumes, and glycogen was extracted by either 30%, 50%, or 72.5% sucrose, boiled or unboiled. Curves represent the mean at a given R_h with the SD being provided on either side of the main line (n = 4-6 with samples having insufficient signal to noise being removed). (A) Glycogen extracted by 30% sucrose, boiled or unboiled; (B) glycogen extracted by 50% sucrose, boiled or unboiled; (C) glycogen extracted by 72.5% sucrose, boiled or unboiled. This figure was adapted from²¹. Abbreviations: SEC = size exclusion chromatography; SD = standard deviation; Rh = hydrodynamic radius; w(log R_h) = SEC weight distribution. Please click here to view a larger version of this figure.

Figure 7: Chain length distributions, N_de(X), of glycogen. Chain length analysis was performed on six livers for both boiled and unboiled using 30%, 50%, and 72.5% sucrose concentrations in the ultracentrifugation step. Values for each DP represent the mean ± SD (N = 6). (A) Glycogen extracted by 30% sucrose, boiled or unboiled; (B) glycogen extracted by 50% sucrose, boiled or unboiled; (C) glycogen extracted by 72.5% sucrose, boiled or unboiled. This figure was adapted from²¹. Abbreviation: N_de(X) = chain length distribution. Please click here to view a larger version of this figure.

	Crude yield (%)	Purity (%)	Glycogen yield (%)
30% Sucrose-c	2.1 ± 1.0^a	13.1 ± 12.0^b	10.7 ± 9.1^a
50% Sucrose-c	1.2 ± 0.7^a	23.3 ± 20.1^b	10.2 ± 8.1^a
72.5% Sucrose-c	1.9 ± 0.8^a	9.8 ± 9.0^b	5.3 ± 2.4^a
30% Sucrose-b	0.8 ± 0.4^a	67.9 ± 16.8^a	16.0 ± 5.1^a
50% Sucrose-b	1.1 ± 0.6^a	48.6 ± 16.9^a	14.8 ± 7.6^a
72.5% Sucrose-b	2.0 ± 0.9^a	14.7 ± 12.6^b	6.9 ± 3.7^a
Two-way ANOVA	Sucrose: P = 0.053	Sucrose: P < 0.001	Sucrose: P = 0.034
	Temperature: P = 0.108	Temperature: P < 0.0001	Temperature: P = 0.116
	Interaction: P = 0.11	Interaction: P = 0.003	Interaction: P = 0.801

Table 1: Purity, crude yield, glycogen yield. Crude yield, purity and glycogen yield for liver glycogen samples extracted by 30%, 50%, or 72.5% sucrose, boiled or unboiled. -c were samples extracted by cold buffer; -b were samples extracted by boiling for 10 min. The values are given as the mean ± standard deviation (SD), n = 6. Differences in values with different superscript letters in the same column are statistically significant (P < 0.05). This table was reproduced with permission from²¹.

		Mean R_h,max	β content	Mean ACL
30% Sucrose-c	29.4 ± 1.5^b	34.9 ± 0.6^a	40.9 ± 6.2%^a,b	4.8 ± 0.5^c
50% Sucrose-c	32.0 ± 1.1^a,b	36.1 ± 0.5^a	28.5 ± 3.0%^b,c	5.6 ± 1.1^b,c
72.5% Sucrose-c	34.3 ± 1.8^a	36.2 ± 0.4^a	23.7 ± 3.5%^c	4.7 ± 0.9^c
30% Sucrose-b	29.4 ± 1.2^b	33.7 ± 3.1^a	43.1 ± 5.1%^a	8.6 ± 1.8^a
50% Sucrose-b	30.1 ± 1.2^b	34.9 ± 0.7^a	36.0 ± 7.2%^a,b,c	8.8 ± 1.7^a
72.5% Sucrose-b	30.9 ± 3.5^a,b	33.6 ± 3.5^a	34.0 ± 13.1%^a,b,c	7.6 ± 1.9^a,b
Two-way ANOVA	Sucrose: P = 0.002	Sucrose: P = 0.442	Sucrose: P < 0.001	Sucrose: P = 0.290
	Temperature: P = 0.010	Temperature: P = 0.032	Temperature: P = 0.016	Temperature: P < 0.0001
	Interaction: P = 0.431	Interaction: P = 0.640	Interaction: P = 0.441	Interaction: P = 0.750

Table 2: and R_h at which the maxima occurs (R_h,max) and average chain length (ACL). and R_h at which the maxima occurs (R_h,max), β particle content, and average chain length (ACL) of glycogen extracted by either 30%, 50%, or 72.5% sucrose, boiled or unboiled. -c unboiled; -b involved a 10 min boiling step. Differences in values with different superscript letters in the same column are statistically significant (P < 0.05). This table was reproduced with permission from ²¹. Abbreviations: R_h = hydrodynamic radius; = ; ACL = average chain length; R_h,max = R_h at which maxima occurs.

Discussion

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 cold acid. While effective in separating the glycogen from other tissue components, these methods are chemically harsh and have been shown to degrade the molecular structure of glycogen²⁷.

A relatively gentle method has since been developed that uses sucrose density gradient centrifugation¹⁷^,¹⁸, allowing glycogen to form in the pellet while most of the cell material remains in the supernatant. This method is particularly useful for liver tissue, with the glycogen α particles being sensitive to acid hydrolysis²⁸. This milder method does, however, have at least two potential mechanisms for the isolation of glycogen diverging in structure from that seen in vivo: 1) smaller, less dense glycogen particles are more susceptible to being left in the supernatant during sucrose density gradient centrifugation¹⁷^,¹⁸, as they may be unable to reach the pellet; 2) The milder conditions may allow glycogen degradation enzymes, which would be denatured in the harsher alkaline/acid extraction conditions, to continue to degrade glycogen particles during extraction.

A recent publication²¹ aimed to help resolve these potential issues by testing a series of sucrose concentrations (and therefore densities), finding that using a concentration of 30%, as opposed to the traditionally used 72.5%, helped minimize the loss of smaller glycogen particles. Future experiments could test even lower concentrations to see if some smaller particles are still preferentially lost in the supernatant during centrifugation. This publication also tested the efficacy of introducing a 10 min boiling step directly after tissue homogenization to denature the glycogen degradation enzymes, thereby preserving the structure of glycogen. It was shown that this step helped inhibit glycogen degradation, with the glycogen chain lengths being significantly preserved. Further experiments in this study provided evidence that this 10 min boiling step was unlikely to cause significant damage to the glycogen structure. However, this boiling step may influence the structure of glycogen-associated proteins, potentially resulting in the denaturation and subsequent dissociation of proteins from the glycogen. Therefore, if proteomics is of interest, using the low sucrose concentration (30%) without boiling (samples kept on ice) might be preferable, with the caveat that the glycogen may be slightly degraded.

When using sucrose density gradient centrifugation without further optimization experiments, the most suitable method is to utilize a relatively low concentration of sucrose (30%) with the introduction of a 10 min boiling step directly after tissue homogenization. There are some limitations to this technique. First, this was optimized for liver glycogen, and it is important to note that it may not be as appropriate for glycogen from other tissues. Second, as mentioned above, the lowest sucrose concentration tested was 30%, and it is possible that lower concentrations could be preferable. Third, an optimized technique that prevents the enzymatic degradation of glycogen while preserving the associated proteome is not yet available.

開示

The authors have nothing to disclose.

Acknowledgements

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.

Materials

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

参考文献

Hills, D. M., Heller, H. C., Hacker, S. D., Hall, D. W., Sadava, D., Laskowski, M., Freeeman, W. H. . Life: the science of biology. 12th edn. , (2020).
Calder, P. C., Geddes, R. Glycogen of high molecular weight from mammalian muscle. Carbohydrate Research. 135 (2), 249-256 (1985).
Rybicka, K. K. Glycosomes – The organelles of glycogen metabolism. Tissue and Cell. 28 (3), 253-265 (1996).
Takeuchi, T., Iwamasa, T., Miyayama, H. Ultrafine structure of glycogen macromolecules in mammalian-tissues. Journal of Electron Microscopy. 27 (1), 31-38 (1978).
Drochmans, P. Morphologie du glycogene – etude au microscope electronique de colorations negatives du glycogene particulaire. Journal of Ultrastructure Research. 6, 141-163 (1962).
Besford, Q. A., et al. The structure of cardiac glycogen in healthy mice. International Journal of Biological Macromolecules. 51 (5), 887-891 (2012).
Lumsden, R. D. Macromolecular structure of glycogen in some cyclophyllidean and trypanorhynch cestodes. Journal of Parasitology. 51 (4), 501-515 (1965).
Deng, B., et al. Molecular structure of glycogen in diabetic liver. Glycoconjugate Journal. 32 (3-4), 113-118 (2015).
Deng, B., et al. The mechanism for stopping chain and total-molecule growth in complex branched polymers, exemplified by glycogen. Biomacromolecules. 16 (6), 1870-1872 (2015).
Jiang, X., et al. The molecular-size dependence of glycogen enzymatic degradation rate and its importance for diabetes. European Polymer Journal. 82 (1), 175-180 (2016).
Hu, Z., et al. Glycogen structure in type 1 diabetic mice: towards understanding the origin of diabetic glycogen molecular fragility. International Journal of Biological Macromolecules. 128, 665-672 (2019).
Suzuki, Y., et al. Insulin control of glycogen metabolism in knockout mice lacking the muscle-specific protein phosphatase PP1G/R-GL. Molecular and Cellular Biology. 21 (8), 2683-2694 (2001).
Stetten, M. R., Katzen, H. M., Stetten, D. Metabolic inhomogeneity of glycogen as a function of molecular weight. Journal of Biological Chemistry. 122 (2), 587-599 (1956).
Nahorski, S. R., Rogers, K. J. An enzymic fluorometric micro method for determination of glycogen. Analytical Biochemistry. 49 (2), 492-497 (1972).
Wang, Z., et al. Molecular structural features of glycogen in the kidneys of diabetic rats. Carbohydrate Polymers. 229, 115526 (2020).
Wang, L., et al. Molecular structure of glycogen in Escherichia coli. Biomacromolecules. 20 (7), 2821-2829 (2019).
Parker, G. J., Koay, A., Gilbert-Wilson, R., Waddington, L. J., Stapleton, D. AMP-activated protein kinase does not associate with glycogen alpha-particles from rat liver. Biochemical and Biophysical Research Communications. 362, 811-815 (2007).
Ryu, J. -. H., et al. Comparative structural analyses of purified glycogen particles from rat liver, human skeletal muscle and commercial preparations. International Journal of Biological Macromolecules. 45 (5), 478-482 (2009).
Sullivan, M. A., et al. Nature of alpha and beta particles in glycogen using molecular size distributions. Biomacromolecules. 11 (4), 1094-1100 (2010).
Tan, X., et al. A new non-degradative method to purify glycogen. Carbohydrate Polymers. 147 (1), 165-170 (2016).
Wang, Z., Liu, Q., Wang, L., Gilbert, R. G., Sullivan, M. A. Optimization of liver glycogen extraction when considering molecular fine structure. Carbohydrate Polymers. 261, 117887 (2020).
Zhao, Y., Tan, X., Wu, G., Gilbert, R. G. Using molecular fine structure to identify optimal methods of extracting starch. Starch – Starke. 72, 1900214 (2020).
Shokri-Afra, H., Ostovar-Ravari, A., Rasouli, M. Improvement of the classical assay method for liver glycogen fractions: ASG is the main and metabolic active fraction. European Review for Medical and Pharmacological Sciences. 20, 4328-4336 (2016).
Kerly, M. The solubility of glycogen. The Biochemical Journal. 24, 67-76 (1930).
Sullivan, M. A., et al. Improving size-exclusion chromatography for glycogen. Journal of Chromatography A. 1332 (1), 21-29 (2014).
Sullivan, M. A., et al. Skeletal muscle glycogen chain length correlates with insolubility in mouse models of polyglucosan-associated neurodegenerative diseases. Cell Reports. 27 (5), 1334-1344 (2019).
Orrell, S. A., Bueding, E. A comparison of products obtained by various procedures used for the extraction of glycogen. Journal of Biological Chemistry. 239 (12), 4021-4026 (1964).
Sullivan, M. A., et al. Molecular insights into glycogen alpha-particle formation. Biomacromolecules. 13 (11), 3805-3813 (2012).

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Wang, Z., Liu, Q., Wang, L., Gilbert, R. G., Sullivan, M. A. The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination. J. Vis. Exp. (180), e63088, doi:10.3791/63088 (2022).

The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination