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 molecules^3,4; 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 particle^8,9,10,11.

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 extent^15,16. 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 ultracentrifugation^17,18,19. 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.