Stores of readily available glucose to supply the tissues with an oxidizable energy source are found principally in the liver, as glycogen. Glycogen is a polymer of glucose residues linked by α-(1,4)- and α-(1,6)-glycosidic bonds. A second major source of stored glucose is the glycogen of skeletal muscle. However, muscle glycogen is not generally available to other tissues, because muscle lacks the enzyme glucose-6-phosphatase.

Glycogen metabolism(Prof. Sylvester L.B. Kajuna)

Glycogen Structure. Section of a glycogen polymer depicting glucose monomers as colored balls. The blue balls represent glucose linked by α1,4 glycosidic bonds. The red balls represent glucose at branch points where there are both α1,4 and α1,6 glycosidic bonds.

The major site of daily glucose consumption (75%) is the brain via aerobic pathways. Most of the remainder of is utilized by erythrocytes, skeletal muscle, and heart muscle. The body obtains glucose either directly from the diet or from amino acids and lactate via gluconeogenesis. Glucose obtained from these two primary sources either remains soluble in the body fluids or is stored in a polymeric form, glycogen. Glycogen is considered the principal storage form of glucose and is found mainly in liver and muscle, with kidney and intestines adding minor storage sites.

With up to 10% of its weight as glycogen, the liver has the highest specific content of any body tissue. Muscle has a much lower amount of glycogen per unit mass of tissue, but since the total mass of muscle is so much greater than that of liver, total glycogen stored in muscle is about twice that of liver. Stores of glycogen in the liver are considered the main buffer of blood glucose levels.

Glycogen homeostasis involves the concerted regulation of the rate of glycogen synthesis (glycogenesis) and the rate of glycogen breakdown (glycogenolysis). Theses two processes are reciprocally regulated such that hormones that stimulate glycogenolysis (e.g. glucagon, cortisol, epinephrine, norepinephrine) simultaneously inhibit glycogenesis. Conversely, insulin, which directs the body to store excess carbon for future use, stimulates glycogenesis while simultaneously inhibiting glycogenolysis.

Degradation of stored glycogen, termed glycogenolysis, occurs through the action of glycogen phosphorylase. There are three distinct human genes encoding proteins with glycogen phosphorylase activity. One gene (PYGL) expresses the hepatic form of the enzyme, a second (PYGM) expresses the muscle form, and the third (PYGB) expresses the brain form.

Glycogenolysis

The action of phosphorylase is to phosphorolytically remove single glucose residues from α-(1,4)-linkages within the glycogen molecules. The product of this reaction is glucose-1-phosphate. The advantage of the reaction proceeding through a phosphorolytic step is that:

1. The glucose is removed from glycogen in an activated state, i.e. phosphorylated and this occurs without ATP hydrolysis.

2. The concentration of Pi in the cell is high enough to drive the equilibrium of the reaction in the favorable direction since the free energy change of the standard state reaction is positive.

Phosphorylase reaction

The glucose-1-phosphate produced by the action of phosphorylase is converted to glucose-6-phosphate by phosphoglucomutase (phosphohexose mutase): this enzyme, like phosphoglycerate mutase of glycolysis, contains a phosphorylated amino acid in the active site (in the case of phosphoglucomutase it is a Ser residue). The enzyme phosphate is transferred to C-6 of glucose-1-phosphate generating glucose-1,6-phosphate as an intermediate.

phosphate on C-1 is then transferred to the enzyme regenerating active enzyme and glucose-6-phosphate is the released product. There are four different phosphoglucomutase genes in humans identified as PGM1, PGM2, PGM3, and PGM5.

The protein encoded by the PGM5 gene is called phosphoglucomutase-like protein 5. The PGM1 gene is expressed in most tissues, whereas PGM2 expression predominates in red blood cells. The PGM1 gene is located on chromsome 1p31 and is composed of 13 exons that generates three alternatively spliced mRNAs and three isoforms of this enzyme. Mutations in the PGM1 gene are associated with the congenital disorder of glycosylation, CDG1T (once referred to as glycogen storage disease type 14, GSD14).

The phosphorylase mediated release of glucose from glycogen yields a charged glucose residue without the need for hydrolysis of ATP. An additional necessity of releasing phosphorylated glucose from glycogen ensures that the glucose residues do not freely diffuse from the cell. In the case of muscle cells this is acutely apparent since the purpose in glycogenolysis in muscle cells is to generate substrate for glycolysis.

The conversion of glucose-6-phosphate to glucose, which occurs in the liver, kidney and intestine, by the action of glucose-6-phosphatase does not occur in skeletal muscle as these cells lack this enzyme. Therefore, any glucose released from glycogen stores of muscle will be oxidized in the glycolytic pathway. In the liver the action of glucose-6-phosphatase allows glycogenolysis to generate free glucose for maintaining blood glucose levels.

Glycogen phosphorylase cannot remove glucose residues from the branch points (α-1,6 linkages) in glycogen. The activity of phosphorylase ceases approximately four glucose residues from the branch point. The removal of the these branch point glucose residues requires the action of glycogen debranching enzyme (GDE). The official name of GDE is amylo-1,6-glucosidase, 4-α-glucanotransferase (gene symbol: AGL) which contains 2 activities: glucotransferase and glucosidase. The AGL gene is located on chromosome 1p21 and is composed of 36 exons that generates three alternative spliced isoform of the enzyme. Isoform 1 contains 1532 amino acids, isoform 2 contains 1515 amino acids, and isoform 3 contains 1516 amino acids.

Glycogen Debranching

The transferase activity of debranching enzyme removes the terminal three glucose residues of one branch and attaches them to a free C-4 end of a second branch. The glucose in α-(1,6)-linkage at the branch is then removed by the action of glucosidase. This glucose residue is uncharged since the glucosidase-catalyzed reaction is not phosphorylytic. This means that theoretically glycogenolysis occurring in skeletal muscle could generate free glucose which could enter the blood stream. However, the activity of hexokinase in muscle is so high that any free glucose is immediately phosphorylated and enters the glycolytic pathway. Indeed, the precise reason for the temporary appearance of the free glucose from glycogen is the need of the skeletal muscle cell to generate energy from glucose oxidation, thereby, precluding any chance of the glucose entering the blood.

 

  

Glycogen debranching activity

For de novo glycogen synthesis to proceed the first glucose residue is attached to a protein known as glycogenin. Glycogenin has the unusual property of catalyzing its own glycosylation, attaching C-1 of a UDP-glucose to a tyrosine residue on the enzyme. The attached glucose then serves as the primer required by glycogen synthase to attach additional glucose molecules via the mechanism described below. There are two glycogenin genes in humans identified as GYG1 and GYG2. The GYG1 gene is located on chromosome 3q24-q25.1 and is composed of 8 exons that generate three splice variant mRNA. These three mRNAs produce three glycogenin-1 isoforms identified as isoform 1 (350 amino acids), isoform 2 (333 amino acids), and isoform 3 (279 amino acids).

Glycogen Synthesis

The GYG1 gene is predominantly expressed in muscle but is also expressed in many other tissues as well. Mutations in the GYG1 gene are associated the recently (2010) characterized glycogen storage disease identified as type 15 (GSD15). The GYG2 gene is located on chromosome Xp22.3 and is composed of 14 exons that multiple splice variant mRNAs leading to the generation of multiple glycogenin-2 isoforms. The GYG2 gene is predominantly expressed in the liver.

Synthesis of glycogen from glucose is carried out by the enzyme glycogen synthase (GS). This enzyme utilizes UDP-glucose as one substrate and the non-reducing end of glycogen as another. The activation of glucose to be used for glycogen synthesis is carried out by the enzyme UDP-glucose pyrophosphorylase. This enzyme exchanges the phosphate on C-1 of glucose-1-phosphate for UDP. The energy of the phospho-glycosyl bond of UDP-glucose is utilized by glycogen synthase to catalyze the incorporation of glucose into glycogen. UDP is subsequently released from the enzyme.

There are two distinct glycogen synthase enzymes in humans. One is expressed in skeletal muscle the other in the liver. The muscle enzyme is encoded by the GYS1 gene and the liver enzyme is encoded by the GYS2 gene. The GYS1 gene is located on chromosome 19q13.3 and is composed of 16 exons that produce two splice variant encoding two isoforms of the muscle enzyme. Isoform 1 is composed of 737 amino acids and isoform 2 is composed of 673 amino acids. The GYS2 gene is located on chromosome 12p12.2 and is composed of 20 exons that produce a protein of 703 amino acids.

Beginning with free glucose, several reactions are required to initiate and then produce glycogen polymers. Glucose is first phosphorylated by hexokinases or glucokinase to glucose-6-phosphate (G6P). G6P is then converted to glucose-1-phosphate (G1P) via the action of phosphoglucomutase (PGM). G1P is then "activated" for glycogen synthesis via the addition of uridine nucleotide catalyzed by G1P uridyltransferase. The resultant UDP-glucose can then be used as a substrate for the self-glucosylating reaction of glycogenin, or if pre-exisiting glycogen polymers exist, the UDP-glucose is utilized as the substrate for glycogen synthase.

Reactions of the addition of glucose to glycogen

The α-1,6 branches in glucose are produced by amylo-(1,4 to 1,6)-transglucosidase, also termed the glycogen branching enzyme (gene symbol: GBE1). This enzyme transfers a terminal fragment of 6-7 glucose residues (from a polymer at least 11 glucose residues long) to an internal glucose residue at the C-6 hydroxyl position. The GBE1 gene is located on chromosome 3p12.3 and is composed of 16 exons that encode a protein of 702 amino acids.

Glycogen Branching

Functional glycogen phosphorylase is a homodimeric enzyme that exist in two distinct conformational states: a T (for tense, less active) and R (for relaxed, more active) state. Phosphorylase is capable of binding to glycogen when the enzyme is in the R state. This conformation is enhanced by binding of AMP (allosteric activator) and inhibited by binding of ATP or glucose-6-phosphate (allosteric inhibitors). The enzyme is also subject to covalent modification by phosphorylation as a means of regulating its activity. The relative activity of the un-modified phosphorylase enzyme (given the name phosphorylase-b) is sufficient to generate enough glucose-1-phosphate for entry into glycolysis for the production of sufficient ATP to maintain the normal resting activity of the cell. This is true in both liver and muscle cells.

Regulation of Glycogen Catabolism

PKA is cAMP-dependent protein kinase. PPI-1 is phosphoprotein phosphatase-1 inhibitor. Green arrows denote positive effects on any enzyme is indicated. Red T-lines indicate inhibitory actions. Briefly, phosphorylase b is phosphorylated, and rendered highly active, by phosphorylase kinase, PHK (glycogen synthase-glycogen phosphorylase kinase, GS/GP kinase). Phosphorylase kinase is itself phosphorylated, leading to increased activity, by PKA (itself activated through receptor-mediated mechanisms). PKA also phosphorylates PPI-1 leading to an inhibition of phosphate removal allowing the activated enzymes to remain so longer. Calcium ions can activate phosphorylase kinase even in the absence of the enzyme being phosphorylated. This allows neuromuscular stimulation by acetylcholine to lead to increased glycogenolysis in the absence of receptor stimulation.

Pathways involved in the regulation of glycogen phosphorylase

In response to lowered blood glucose the α cells of the pancreas secrete glucagon which binds to cell surface receptors that are predominantly found on hepatocytes. Glucagon receptors are only found on one other cell type, white adipocytes, but at significantly lower levels than those seen on heptocytes. Because of this distribution of receptors, it is easy to understand why liver cells are the primary target for the action of glucagon. The glucagon receptor is a Gα-coupled GPCR. The response of cells to the binding of glucagon to its cell surface receptor is, therefore, the activation of the enzyme adenylate cyclase.

Activation of adenylate cyclase leads to a large increase in the formation of cAMP which then binds to, and activates the enzyme cAMP-dependent protein kinase, PKA (see Figure below). Binding of cAMP to the regulatory subunits of PKA leads to the release and subsequent activation of the catalytic subunits. The catalytic subunits then phosphorylate a number of proteins on serine and threonine residues.

Pathways involved in the regulation of glycogen synthase by epinephrine: Epinephrine (or norepinephrine) activation of α1-adrenergic receptors results in the activation of PLCβ. See the text for details of the regulatory mechanisms. PKC is protein kinase C. PLCβ is phospholipase Cβ. The substrate for PLCβ is phosphatidylinositol-4,5-bisphosphate (PIP2) and the products are IP3 and DAG.

The net effects of the various phosphorylations of glycogen synthase result in:

1. decreased affinity of synthase for UDP-glucose.

2. decreased affinity of synthase for glucose-6-phosphate.

3. increased affinity of synthase for ATP and Pi.

Reconversion of glycogen synthase-b to glycogen synthase-a requires dephosphorylation. This is carried out predominately by the serine/threonine phosphatase described earlier, PP1. This, of course is the same phosphatase involved in the dephosphorylation of glycogen phosphorylase described above. Although another serine/threonine phosphatase, namely protein phosphatase-2A (PP-2A), has been shown to dephosphorylate glycogen synthase in vitro, its role in vivo is significantly less than that of PP1.

The activity of PP1 is also affected by insulin. The pancreatic hormone exerts an opposing effect to that of glucagon and epinephrine. This should appear obvious since the role of insulin is to increase the uptake of glucose from the blood.

Since glycogen molecules can become enormously large, an inability to degrade glycogen can cause cells to become pathologically engorged; it can also lead to the functional loss of glycogen as a source of cell energy and as a blood glucose buffer. Although glycogen storage diseases are quite rare, their effects can be most dramatic. The debilitating effect of many glycogen storage diseases depends on the severity of the mutation causing the deficiency. In addition, although the glycogen storage diseases are attributed to specific enzyme deficiencies, other events can cause the same characteristic symptoms. For example, Type I glycogen storage disease (von Gierke disease) is attributed to lack of glucose-6-phosphatase.

Glycogen Storage Diseases

However, this enzyme is localized on the cisternal surface of the endoplasmic reticulum (ER); in order to gain access to the phosphatase, glucose-6-phosphate must pass through a specific translocase in the ER membrane (see Figure below). Mutation of either the phosphatase or the translocase makes transfer of liver glycogen to the blood a very limited process. Thus, mutation of either gene leads to symptoms associated with von Gierke disease, which occurs at a rate of about 1 in 200,000 people.

The metabolic consequences of the hepatic glucose-6-phosphate deficiency of von Gierke disease extend well beyond just the obvious hypoglycemia that results from the deficiency in liver being able to deliver free glucose to the blood. The inability to release the phosphate from glucose-6-phopsphate results in diversion into glycolysis and production of pyruvate as well as increased diversion onto the pentose phosphate pathway. The production of excess pyruvate, at levels above of the capacity of the TCA cycle to completely oxidize it, results in its reduction to lactate resulting in lactic acidemia.

Mechanisms of glucose-6-phosphate conversion to free glucose.

In addition, some of the pyruvate is transaminated to alanine leading to hyperalaninemia. Some of the pyruvate will be oxidized to acetyl-CoA which can't be fully oxidized in the TCA cycle and so the acetyl-CoA will end up in the cytosol where it will serve as a substrate for triglyceride and cholesterol synthesis resulting in hyperlipidemia. The oxidation of glucose-6-phophate via the pentose phosphate pathway leads to increased production of ribose-5-phosphate which then activates the de novo synthesis of the purine nucleotides. In excess of the need, these purine nucleotides will ultimately be catabolized to uric acid resulting in hyperuricemia and consequent symptoms of gout. The interrelationships of these metabolic pathways is diagrammed in the Figure below.

In the absence of glucose-6-phosphatase activity free glucose cannot be release from the liver contibuting to severe fasting hypoglycemia. In addition the increased glucose-6-phosphate levels lead to increased pentose phosphate pathway (PPP) activity as well as increased glycolysis to pyruvate. The incresased levels of pyruvate lead to increased lactate produciton via lactate dehydrogenase (LDH) and alanine via alanine transaminase (ALT). In addition, the increased pyruvate is oxidized via the pyruvate dehydrogenase complex (PDHc) leading to increased production of acetyl-CoA which is, in turn, used for the synthesis of fatty acids and cholesterol.

Interrelationships of metabolic pathway disruption in von Gierke disease

The excess glycolysis also results in increased production of glycerol-3-phosphate (G3P) from DHAP via the action of glycerol-3-phosphate dehydrogenase (GPD1). Increased G3P and fatty acids leads to increased triglyceride synthesis which, in conjunction with the increased cholesterol, leads to hyperlipidemia as well as fatty infiltration in hepatocytes contributing to hepatomegaly and cirrhosis.

The glycogen storage diseases are divided into two primary categories: those that result principally from defects in liver glycogen homeostasis and those that represent defects in muscle glycogen homeostasis. The liver glycogen storage diseases result in hepatomegaly and hypoglycemia or cirrhosis, whereas the muscle glycogen storage diseases result in skeletal and cardiac myopathies and/or energy impairment. The most notable muscle glycogen storage disease is Pompe disease (type II GSD).

Several glycogenoses are the result of deficiencies in enzymes of glycolysis whose symptoms and signs are similar to those seen in McArdle disease (type V GSD). These include deficiencies in muscle phosphoglycerate kinase and muscle pyruvate kinase as well as deficiencies in fructose 1,6-bisphosphatase, lactate dehydrogenase and phosphoglycerate mutase.