The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 ....

Protokół

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

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

2. Preparation of buffer and reagents

1. Prepare glycogen isolation buffer containing 5 mM Tris,150 mM NaCl, 2 mM EDTA, ....

Wyniki

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.......

Dyskusje

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.......

Materiały

Name	Company	Catalog Number	Comments
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