What is glycogen and why do we need it?

Glycogen is a branched polymer made up of D-glucose units, the most abundant monosaccharide in nature.
Due to the branched structure, glycogen is a compact and soluble macromolecule, has a low osmotic pressure and allows rapid release of the stored glucose when needed. As it is synthesized without a template, unlike proteins and nucleic acids it exists as a population of molecules with different chemical structures and sizes.
Glycogen molecule forms assemblages with proteins that are essential for its metabolism, such as glycogenin (EC 2.4.1.186), glycogen synthase (EC 2.4.1.11), glycogen phosphorylase (EC 2.4.1.1), debranching enzyme (EC 2.4.1.25 and EC 3.2.1.33), and those that regulate its anchoring to cytoskeleton and membranes. Such polysaccharide-proteins are called beta granules and are found in the cytosol of bacteria, Archea, Fungi and animal cells.
Phosphate groups are covalently linked to the polysaccharide, and, as well as associated proteins, seem to be involved in the regulation of its metabolism.
Glycogen is stored in cell in times of nutritional plenty and serves as storage form of glucose and therefore of energy. In animals, it is found in practically all cells and, in mammals, it is most abundant in the liver and skeletal muscle. In the liver, several beta granules arrange in a broccoli-like fashion to form the so-called alpha granules. Glycogen is also found in lysosomes.
In humans, it represents less than 1% of the body’s energy stores, and is essential in maintaining blood glucose homeostasis, too.
It is absent in plants, where starch is the storage form of glucose. Therefore, the polymerization of glucose represents a universal mechanism for energy storage.
From a human nutrition point of view, glycogen has little significance as after an animal has been killed it is mostly broken down to glucose and then to lactic acid. It should be noted that the acidity consequently to lactic acid production gradually improves the texture and keeping qualities of the meat. The only dietary sources are oysters and other shellfish that are eaten virtually alive; they contain about 5% glycogen.

CONTENTS

The discovery of glycogen

Glycogen was discovered in 1857 by the French physiologist Claude Bernard, considered the founder of experimental medicine. In the second half of the last century, studies on glycogen metabolism led to several significant discoveries, such as:

  • the reversible phosphorylation of proteins;
  • protein kinases and protein phosphatases;
  • the effect of insulin on the activity of intracellular enzymes.

In turn, this led to the award of four Nobel Prizes, three for physiology or medicine, to Carl Ferdinand Cori and Gerty Theresa Cori, née Radnitz in 1947, Earl Sutherland Jr. in 1971, and Edwin Krebs and Edmond Fischer in 1992, and one for chemistry, to Louis Leloir in 1970.

Chemical and molecular structure of glycogen

Individual glycogen molecule is a branched polymer of D-glucose in the pyranose form, namely, a stiff six-membered heterocyclic ring, five carbons and one oxygen, with chair conformation.
The central priming protein glycogenin and phosphate groups are covalently bound to the polysaccharide  chain.
Most of the glucose units are linked by α-1,4 glycosidic bonds, where each unit is linked to the next by a bond between C-1 of one unit and the hydroxyl group on the C-4 of the next unit, with an oxygen atom acting as a bridge between the two carbon atoms.
The branch points are introduced by α-1,6 glycosidic bonds, that occur approximately every 8-12 residues, again with an oxygen atom acting as a bridge between the two carbon atoms, in this case, C-1 and C-6, and with an average chain length of about 13 residues in mammals. Because each branch ends in a non-reducing residue, there are n+1 non-reducing ends in the molecule, where n is number of chains, but only one reducing end to which glycogenin is linked.
Note: in disaccharides, oligosaccharides, and polysaccharide the non-reducing end is the end that lacks a free anomeric carbon atom.

Chemical and molecular structure of glycogen

Having the same types of bonds, the primary structure of glycogen resembles that of amylopectin, which, with amylose, is one of the two polymers of D-glucose units composing starch. However, compared to amylopectin where branches occur every 25-30 glucose units, glycogen is more branched, and the branches are smaller.
Unlike proteins and nucleic acids, polysaccharides are synthesized without a template, resulting from the addition of monosaccharides or oligosaccharides to the growing structure. In addition, because branches occur without a precise localization, molecules with the same mass will not necessarily have the same structure. Hence, for each type of molecules there are different chemical structures. Moreover,  glycogen isolated from different biological sources exists as a population of molecules of different sizes. Therefore, the best way to describe its chemistry is to define the distribution of the molecular masses, and the average frequency with which branches occurs and their average length.
Finally, it should be emphasized that glycogen is not a static entity but constantly vary over the course of its existence.

As glucose is a chiral molecule, it exists as a pair of enantiomers, indicated according to the Fischer-Rosanoff convention as D-glucose, the most widespread in nature and the monomeric unit of glycogen and starch, and L-glucose.

Who does stabilize 3D structure?

The folding into three-dimensional structures of macromolecules such as proteins, nucleic acids and polysaccharides is governed by the same principles: the monomeric units, namely, amino acids, nucleotides, and monosaccharides, with their more-or-less rigid structure, are joined by covalent bonds to form one dimensional polymers that spontaneously fold into three-dimensional structures stabilized by noncovalent interactions such as:

  • hydrogen bonds;
  • van der Waals interactions;
  • hydrophobic interactions;
  • ionic interactions, when charged subunits are present.

These interactions can occur within macromolecules or between macromolecules, as in supramolecular complexes such as cellulose or multienzyme complexes.
As the pyranose ring of glucose is a rigid structure, the three-dimensional conformation of the oligosaccharides and polysaccharides results from rotation about both C−O bonds of the glycosidic bond, with the bond angles labeled φ and ψ. However, there is no free rotation about each C−O bonds due to the steric interference by substituents. Hence, some conformations will be more stable than others. For amylose and glycogen, the most stable 3D structure is a tightly coiled helix stabilized by interchain hydrogen bonds.

What are the advantages of the branched structure?

The highly branched structure of glycogen offer several advantages.

  • The non-reducing ends present on the outermost tier can act as a substrate for glycogen phosphorylase. Therefore, many glycogen phosphorylases can work simultaneously allowing a rapid mobilization of stored glucose as glucose 1-phosphate.
  • The highly branched structure allows stored glucose to exert a much lower osmotic pressure than it would exert if it were in its monomeric form. For example, hepatocytes store an amount of glucose that, in free form, would have a concentration of ~0.4 M, against a glycogen concentration of ~0.01 mM. Therefore, if glucose were in free form, the resulting osmolarity would be so elevated to cause an osmotic entry of water that would lead to cell lysis. Moreover, as extracellular  concentration of glucose is ~5 mM, glucose uptake into a cell with a glucose concentration of 0.4 M would be particularly energetically expensive.
  • Branches allow the formation of compact granules.
  • If branches were absent or at least few, a very large number of long linear polymers would have to have to be present to have a number of non-reducing ends comparable to those present in glycogen and to store a comparable amount of glucose. This could cause cell damage. Evidence in favor of this hypothesis comes from a rare genetic disease, Anderson’s disease or amylopectinosis or glycogen storage disease type 4, due to mutations in the gene for branching enzyme. These mutations lead to a deficiency of the enzyme activity and accumulation in different tissues of abnormally branched glycogen that resembles amylopectin.
  • Branches allow glycogen to remain soluble, unlike starch.

Whelan’s model

Due to the action of glycogenin, and then of glycogen synthase and branching enzyme (EC 2.4.1.18), glycogen molecule grows exponentially in concentric tiers around the glycogenin core. According to Whelan’s model of glycogen structure, two types of glucose chain can be categorized:

  • A-chains, unbranched and present only on the surface;
  • B-chains, internal and, on average, with two branching points.

It has been calculated that the maximum size would be of 12 tiers, for a diameter of ~42 nm, a total number of ~55,000 glucose units, and a molecular mass of ~107 kDa.
Moreover, considering that each tier has a thickness of 3.8 nm, assuming glycogen molecule to be a sphere, from the third to the twelfth tier:

  • the diameter increases by 5.4 times;
  • the volume, which grows according to the cube of its radius, increases 156 times;
  • carbohydrate content increases 45.6 fold;
  • the number of A-chains in each outermost tier increase exponentially, and is equal to 2n-1, where n corresponds to the number of the tier.
Tier Diameter (nm) Chain/Tier Glucose/Tier Total glucose
1 1 13 13
2 3.8 3 26 39
3 7.8 7 52 91
4 11.6 15 104 195
5 15.4 31 208 403
6 19.2 63 416 819
7 23 127 832 1651
8 26.8 255 1664 3315
9 30.6 511 3328 6643
10 34.4 1023 6656 13299
11 38.2 2047 13312 26611
12 42.0 4095 26624 53235

Considering skeletal muscle, the analysis of the size of glycogen molecules by electron microscopy showed the presence of few full-size particles, and an average diameter of ~25 nm, corresponding to seven tiers.
An important feature of the Whelan’s model  is that the outermost tier would contain, in the form of A-chains, ~50% of all of glucose molecules. This does not mean that these molecules are all accessible to glycogen phosphorylase because the enzyme stalls four residues from the branch point. The intervention of the debranching enzyme, whose activity is slower than glycogen phosphorylase activity, removes the branch and allows glycogenolysis to proceed.

Why is 13th tier not possible? The 13th tier seems to be not possible because of the steric hindrance due to high density of glucose units on the molecule surface. Such an high density of glucose residues would lead to insufficient space for the interaction between the catalytic region of glycogen metabolism enzymes, and then of glycogen synthase, too, and the growing chains.
Furthermore, through mathematical analyzes, it has been suggested that values concerning branch length, ~13 residues in mammals, average branching frequencies per tier, 2, and the maximum number of tiers, 12, are optimal for mobilization of the maximum amount of glucose molecules in the shortest possible time.

Glicogenin

The structure of the glycogen molecule includes the protein glycogenin, which is covalently bonded to the polysaccharide chain. Glycogenin initiates the synthesis of glycogen by autoglycosylation, catalyzing the addition of 7-11 glucose units to a specific tyrosine residue. This primer chain then acts as substrate for  glycogen synthase. In addition, binding to actin filaments, glycogenin anchors the oligosaccharide primer chain to the cytoskeleton.

Phosphate groups

In addition to glycogenin, glycogen molecule covalently binds phosphate groups.
For many years they were considered a contaminant and their amounts were inversely correlated with purity of the sample. Only in the early 1980s they were recognized as an integral part of the polysaccharide, where they seems to be linked to C-2 e C-3 as monoester, probably as a result of a side reaction during the activity of glycogen synthase.
Many studies have suggested that their presence plays a role in regulating glycogen metabolism, similarly to what happens for starch metabolism in plants. Evidence supporting this hypothesis are the identification of laforin, a glycogen phosphatases, and that its mutation is a key factor in Lafora disease, a form of epilepsy characterized, among other things, by an excessive phosphorylation of glycogen.
But how would they act? Several hypotheses have been proposed, and two are reported below.

  • It has been suggested that phosphate group, which are hydrophilic, could expose hydrophobic regions, reducing glycogen solubility. The dephosphorylation by laforin, allowing glycogen molecule to remain soluble, would facilitate branch formation.
  • Another hypothesis suggests that the degree of phosphorylation would be related to the age of the molecule, acting as a kind quality control. The increase in phosphorylation, which causes a reduction in the solubility of the polysaccharide, would be considered as a metabolic marker that directs it towards the lysosomal degradation, a process called glycophagy, rather than towards glycogenolysis.

Beta granules

Individual glycogen molecules are too small to be detected by light microscopy. Conversely, electron microscopy allowed to identify three types of structures: beta granules, gamma particles, and alpha granules.
Beta granules are made up of the polysaccharide, glycogenin and gamma-particles, which are protein-rich particles of ~3 nm in diameter. Beta granules have a molecular mass of ~106-107 kDa, a diameter of ~20-30 nm, with a rosette-like appearance.
They are considered a rapid energy source.
Under physiological conditions, proteins account for 66-80% of their weight. These proteins also bind to each other, to cytoskeleton or to membranes, and are all involved in the metabolism of the polysaccharide. Some of these are:

  • glycogen synthase, the debranching enzyme, and glycogen phosphorylase;
  • several regulatory proteins, such as:

laforin and phosphoprotein phosphatase 1 or PP1 (EC 3.1.3.17);

phosphorylase kinase (EC 2.7.11.19) and AMPK (EC 2.7.11.31);

the membrane anchoring protein STDB1; note that phosphorylase kinase binds the membranes, too;

malin or E3-ubiquitin ligase (EC 2.3.2.27), which binds to glycogen via laforin, and TRIM7.

Unlike the pyruvate dehydrogenase complex or ribosomes, the stoichiometry and the composition of the beta granules is not constant, but rather dynamic, as proteins associate or dissociate from the granule depending on cellular conditions. In addition, differences are observed not only between different cell types but also within the same cell type, for example in skeletal muscle cells depending on different subcellular localizations.

Alpha granules

In the liver, beta granules are organized to form structures called alpha granules.
They are made up of several beta granules, are ~108 kDa in molecular weight, up to ~300 nm in diameter, with a broccoli-like appearance.
Alpha granules are considered a slower energy source than beta granules.
To date, the mechanism underlying their formation is not yet clear, although it seems that beta granules are linked through a protein skeleton rich in disulfide bonds.

Where is glycogen found in humans?

In humans, glycogen occurs in all cells, although the main stores are found in the liver and skeletal muscle, where, depending on nutritional status, it can represent up to 10% of the liver mass and 2% of muscle mass. Therefore, skeletal muscle has a limited capacity to store glycogen than liver; however, as its mass is greater than that of the liver, the muscle content of glycogen is about double than that of the liver. For example, in a non-fasting 70 kg adult male there are ~100 g of glycogen in the liver and ~250 g in the muscle. Athletes can reach higher values, as in best male marathon runners, whose stores in liver and muscle are equal to ~475 g, corresponding to ~1,900 kcal.
The amount of glycogen stored are much lower than those of fats because fats are a form of energy storage much more efficient. Why?

  • They can be stored in anhydrous form, whereas the amount of water bound to glycogen is equal to 2-3 times its weight.
  • As the stored fats are insoluble in water they are osmotically inert;
  • The oxidation of one gram of glycogen yield about 4 kcal, whereas one gram of fat about 9 kcal, then about double the energy.

Why is glycogen important to humans?

Fats, proteins and glycogen are energy stores that the body uses when needed.
In animals, fats are second only to proteins as reserve of energy, although proteins are an energy store of last resort, such as during a prolonged fast.
In a healthy adult subject, fat mass accounts for about 21% of the body weight in man and 26% in woman. In an adult male of 70 kg body weight, fat mass is sufficient for about 2 months of the body’s energy expenditures. Conversely, glycogen stores are sufficient for about one day of the body’s energy expenditures. Nevertheless, glycogen is accumulated. Why?

  • Unlike glucose, fatty acids, a class of lipids cannot be metabolized anaerobically and therefore cannot be used to produce energy by skeletal muscle during anaerobic exercises. Moreover, muscle cannot oxidize fatty acids as quickly as it does with glucose stored in glycogen.
  • Animals cannot convert fatty acids into glucose, so they cannot be used to maintain glycemic homeostasis. Although glucose released from muscle glycogen remains within the cell, glucose released from hepatic glycogen, and to a lesser extent from renal glycogen, thanks to the enzyme glucose 6-phosphatase, enter the systemic circulation contributing to the regulation of blood glucose levels.
  • Glycogen has a specialized role in fetal lung type II pulmonary cells, or type II pneumocytes, the lung cells  susceptible to SARS-CoV-2. At about 26 weeks of gestation they start to accumulate glycogen that serves as major substrate for the synthesis of the lipids of pulmonary surfactant, of which dipalmitoylphosphatidylcholine is the major component.
  • The brain contains a small amount of glycogen, too, primarily in astrocytes. It accumulates during sleep and is mobilized upon waking, therefore suggesting its functional role in the conscious brain. These glycogen stores also provide a moderate degree of protection against hypoglycemia.

Glycogen and muscle work

Carbohydrates, namely glucose, and fatty acids are the main energy sources for muscle during exercise, and their relative contribution varies depending on the intensity and duration of exercise, as summarized below:

  • <30% VO2max: mainly fatty acids;
  • 40-60% VO2max: fatty acids and carbohydrates;
  • 75% VO2max: mainly carbohydrates;
  • >80% VO2max: almost exclusively carbohydrates.

Therefore, the contribution of glycogen to the energy needed to support muscle work increases with increasing exercise intensity, whereas that of fatty acids decreases. Furthermore, when no carbohydrates are ingested, performance is determined by glycogen stores in skeletal muscle and liver, whose relative consumption is different: as the intensity increases, muscle glycogen consumption increases whereas liver glycogen consumption remains more or less constant.

Note: the relative contribution of fatty acids and glycogen as energy sources also varies according to the athlete’s level of training.

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