Glycogen metabolism

Subtopics

 Introduction  Degradation of glycogen: glycogen phosphorylase, glycogen debranching enzyme, phosphoglucomutase and glucose-6 phosphatase  Glycogen synthesis: UDP-glucose pyrophosphorylase, glycogenin, glycogen synthase, glycogen branching enzyme and significance of glycogen branching  Thermodynamics of glycogen metabolism  Control of glycogen metabolism: allosteric regulation and bicyclic cascade of glycogen phosphorylase and glycogen synthase  Integration of glycogen metabolism: cAMP-dependent phosphorylation cascade (G-proteins, adenylate cyclase, protein kinase A, phosphorylase kinase) and calcium-dependent phosphorylation cascade (Calmodulin and phosphorylase kinase)  Signal amplification and signal termination by phosphoprotein phosphatase 1  Insulin and the well-fed state, maintenance of blood glucose level and glucose transporters (GLUT1-GLUT5)  Glucagon and the fasting state  Epinephrine and the stress response, the fructose-2,6-bisphosphate control system  Glycogen storage diseases: Deficiencies of G6Pase, glucosidase, debranching enzyme, branching enzyme and muscle phosphorylase.

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Glycogen metabolism

Introduction

Glucose cannot be stored because high concentrations of glucose disrupt the osmotic balance of the cell. Higher organisms protect themselves from potential fuel shortage and osmotic damage by polymerizing excess glucose into glycogen. Glycogen is a high molecular weight storage polymer of glucose that can be readily mobilized in times of metabolic demand. It is predominantly α (1→4)-linked glucose polymer with α (1→6) branches every 8 to 14 residues. Glycogen is stored as large cytoplasmic particles in the form of hydrated granules. These cytoplasmic granular particles contain the enzymes that metabolize glycogen, as well as the machinery for regulating these enzymes. Glycogen forms a left- handed helix with 6.5 glucose residues per turn, similar to α-amylose.

No matter how big, glycogen has but one reducing end. Glucose units from glycogen are mobilized by their sequential removal from their nonreducing ends. Storing energy as glucose polymers is common to all forms of life. It is present in eukaryotes, bacteria and archae. In plants, the glucose storage substance is starch. Glucose released from glycogen, unlike triacylglycerols from lipids, can be metabolized in the absence of oxygen and thus it can supply energy for anaerobic activity. Glycogen is a preferred source of energy for sudden, strenuous activity of muscles which cannot mobilize fats as rapidly as glycogen. The fatty acid residues of lipids cannot also be catabolized anaerobically. Furthermore, animals cannot convert fatty acids to glucose so fat metabolism alone cannot adequately maintain essential blood glucose levels.

The two major storage tissues for glycogen storage in animals are the liver and skeletal muscles. Liver glycogen serves as a reservoir of glucose for other tissues when dietary glucose is not available. It maintains glucose homeostasis of the organism as a whole. In mammals, the neurons of brain have virtually no stored source of energy and they cannot use fatty acids as fuel. They are rather completely dependent on a constant supply of glucose from the blood. The circulating blood keeps the brain supplied with glucose, which is virtually the only fuel used by the brain, except during prolonged starvation. Hence glycogen in the liver is especially important to maintain the blood-glucose levels as required to meet the needs of the organism as a whole. In contrast, glycogen in muscle is utilized to meet the energy needs of only the selfish muscle. Muscle glycogen serves as a reservoir for muscle cells to provide a quick source of energy for either aerobic or anaerobic metabolism. Muscle contraction is driven by generating ATP through glycolysis of glucose. Muscle glycogen can be exhausted in less than

2 | P a g e an hour during vigorous activity. The enzymes in liver and muscle differ in subtle yet important ways that reflect the different roles of glycogen in the two tissues.

Glycogen breakdown

Glycogenolysis or glycogen breakdown is a catabolic pathway from glycogen to glucose-6-phosphate. It provides the energy essential to oppose the forces of entropy and biosynthetic precursors. Glycogen degradation comprises three steps catalyzed by three enzymes: the release of glucose-1-phosphate from glycogen by glycogen phosphorylase, glycogen remodeling by glycogen debranching enzyme and the conversion of glucose-1-phosphate (G1P) into glucose-6-phosphate (G6P) by phosphoglucomutase. Glycogen phosphorylase (or phosphorylase) catalyzes the sequential removal of glucosyl residues from the nonreducing ends of the glycogen substrate by the addition of orthophosphate (Pi) to yield G1P. It hydrolyzes α (1→4)-linked glucosyl units. The cleavage of a bond through the addition of a phosphate group is referred as phosphorolysis or phosphorolytic cleavage.

Phosphorylase can only release a glucose unit that is at least five units from a branch point. Two activities are required to remodel glycogen by converting the branched structure into a linear form and permit further degradation by glycogen phosphorylase. These are a transferase and α-1,6-glucosidase activities. Glycogen debranching enzyme removes glycogen’s branches to allow the phosphorylase reaction to go to completion. It catalyzes both the transfer of three-residue chains onto the nonreducing ends of other chains and the hydrolysis of the remaining α(1→6)-linked glucosyl unit to yield glucose. The cleavage of a bond by addition of water is called hydrolysis or hydrolytic cleavage. Phosphorolysis of glycosidic bonds differs from hydrolysis by amylase during intestinal degradation of dietary glycogen and starch. In phosphorolysis, some of the energy of the glycosidic bond is preserved in the formation of the phosphate ester in G1P. G1P released from glycogen breakdown can be readily converted into G6P by phosphoglucomutase. G6P is suitable form for further metabolism.

A. Glycogen phosphorylase

Glycogen phosphorylase catalyzes the cleavage of an α(1→4) glycosidic bond between two glucose residues at any nonreducing end of glycogen. The reaction removes the terminal glucose residue as α-D- G1P through attack by inorganic phosphate (Pi). It catalyzes the controlling step in glycogen breakdown. Phosphorylase is a dimer of two identical subunits localized in the cytosol. Each subunit is compactly folded into an amino-terminal domain with a glycogen-binding site and a carboxyl-terminal domain. Phosphorylase is regulated both by both allosteric interactions and post-translational modification. Each

3 | P a g e subunits of the phosphorylase dimer can interconvert between a less active tensed (T) state and a more active relaxed (R) state. The T state is less active because the catalytic site is partly blocked by a loop. Allosteric inhibitors of phosphorylase such as ATP, G6P, and glucose favor the formation of the T state whereas allosteric activator such AMP favors the formation of the R-state. Covalent modification by phosphorylation on Ser-14 of each subunit favors the structure of the R state.

Phosphorylase exists in two types of interchangeable conformers: phosphorylase b (catalytically inactive) and phosphorylase a (catalytically active). Both forms undergo equilibrium between the T and R states in which the unmodified form exist predominantly in the T state whereas the phosphorylated form exists predominantly in the R state. Allosteric regulators interact differently with the phospho- and dephosphoenzymes. Moreover, there are two isozymic forms of glycogen phosphorylase encoded by different genes: one specific to liver and one specific to skeletal muscle. In human beings, the liver and muscle forms are approximately 90% identical in amino acid sequence, yet the 10% difference results in subtle but important shifts in the stability of different forms of the enzyme. The a-form in liver can be reverted to the low-activity T state upon binding of glucose to its active site. The a-form in liver functions as glucose sensor. On the other hand, the b form in muscle can be activated by the binding of AMP, an effect counteracted by ATP and glucose 6-phosphate. Glycogen phosphorylase provides a clear example of the use of enzymatic isoforms to establish tissue-specific regulatory properties.

The special challenge in the glycogen phosphorylase reaction is the exclusion of water from the active site. To this end, phosphorylase contains pyridoxal-5’-phosphate (PLP) group which functions as an essential cofactor. PLP is a derivative of pyridoxine (vitamin B 6). The aldehyde group of PLP forms a Schiff-base linkage with a Lys 680 of the enzyme. It is similarly linked to a variety of enzymes involved in transamination reactions. A Schiff base, also called an imine, is formed by the reaction of a primary amine with an aldehyde or ketone to produce a carbon–nitrogen double bond. The glycogen-binding site of phosphorylase is 30 Å away from the catalytic site on the surface which is connected to the catalytic site by a narrow crevice that can accommodate four or five sugar residues in a chain. However, this crevice is too narrow to admit branched oligosaccharides. The large separation between the substrate binding site and the catalytic site allows the enzyme to cleave many residues before the enzyme must bind the glycogen substrate again. An enzyme that can catalyze many reactions without having to dissociate and reassociate after each catalytic cycle is said to be processive. Processivity is a property of enzymes that metabolize large polymers.

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The catalytic mechanism of the phosphorolytic cleavage of glycogen requires the participation of the phosphate group of PLP as a general acid–base catalyst. The phosphoryl group of PLP is within hydrogen bonding distance of the orthophosphate (Pi). The 5’ phosphate group of PLP acts in tandem with orthophosphate by serving as a proton donor and then as a proton acceptor. Bond cleavage is facilitated by a kind of cyclic proton relay from PLP to Pi and back to PLP. Both the glycogen substrate and the G1P product have the same configuration about C-1 suggesting that the reaction occurs with the overall retention of configuration. There are two alternative mechanisms proposed for glycogen phosphorylase. These are the carbonium ion and the oxonium ion mechanisms. In the first step in both proposed mechanisms, the reacting orthophosphate (Pi) group occupies a position between the 5’-phosphate group of PLP and the glycogen substrate form E─Pi─glycogen ternary complex.

In the second step, the protonated 5’-phosphate group of the bound PLP readily donates a proton to the Pi allowing the latter to donate its proton to the C-4 oxygen forming the α-1,4 glycosidic linkage. The second step is essentially acid catalysis by Pi to generate a carbocation intermediate from the departing glucose residue. Alternatively, acid catalysis facilitated by proton transfer from PLP can form a shielded temporary oxonium ion intermediate with half-chair conformation. In this mechanism, the second step generates α-linked terminal glucosyl residue. In the third step, the carbonium ion or the oxonium ion and Pi combine to form α-D-G1P with the concomitant return of a proton to pyridoxal phosphate. The 1,5-gluconolactone is a potent inhibitor of phosphorylase suggesting that it is a transition state analog that mimics the oxonium ion at the active site of phosphorylase. The shielded oxonium ion intermediate is similar to the transition state in the lysozyme reaction which also involves glycosidic bond cleavage in a polysaccharide).

Analyses of the primary structures of glycogen phosphorylase from E. coli, yeast, slime mold, chlamydomonas, potatoes, rats and human beings have enabled inferences that the catalytic mechanism of glycogen phosphorylase has been maintained throughout evolution. The 16 residues that come into contact with glucose at the active site are nearly identical. The 15 residues at the pyridoxal phosphate-binding site are also substantially conserved. Moreover, the glycogen-binding site is well conserved in all the enzymes. Differences arise, however, when the regulatory sites are compared. The regulation of glycogen phosphorylase became more sophisticated as the enzyme evolved. The simplest type of regulation is feedback inhibition by G6P. Indeed, G6P regulatory site is highly conserved among most of the phosphorylases. In contrast, the crucial amino acid residues that participate in regulation by

5 | P a g e phosphorylation and nucleotide binding are well conserved only in the mammalian enzymes. Thus, this level of regulation was a later evolutionary acquisition.

B. glycogen debranching enzyme

Glycogen breakdown occurs in steps. Firstly, the α-1,4-glycosidic bonds on each branch are cleaved by phosphorylase to yield G1P, leaving four residues along each branch. Secondly, the α(1→4)-linked trisaccharide unit from a “limit branch” of glycogen is transferred to the nonreducing end of another branch by α(1→4) glycosyl transferase. Thirdly, the α(1→6) bond of the residue remaining at the branch point is hydrolyzed by α-1,6-glucosidase to yield free glucose. Phosphorylase stops cleaving α-1,4 linkages when it reaches a terminal residue four glucose residues away from α(1→6) branch point. Continued degradation by the phosphorylase requires glycogen remodeling by glycogen debranching enzyme. Debranching has two components: breaking and reforming α(1→4)-glycosidic bonds and the hydrolysis of α(1→6)-glycosidic bonds. About 90% of the residues in glycogen are phosphorolytically cleaved by phosphorylase whereas 10% of the residues in glycogen are hydrolytically cleaved by α-1,6- glucosidase.

During glycosyl transferase reaction, the old α-1,4-glycosidic link is broken and a new α-1,4 link is formed. The transferase enzyme is formally known as oligo α(1→6) to α(1→6) glucantransferase. It shifts a block of three glucosyl residues from one outer branch to the other exposing a single glucose residue joined by an α-1,6-glycosidic bond. The branching residue is hydrolyzed by α-1,6-glucosidase leaving a linear chain. Once these branches are removed by glycogen remodeling enzymes, the newly elongated linear chain is subject to degradation by glycogen phosphorylase. In eukaryotes, the transferase and the α-1,6-glucosidase activities of the debranching enzyme are present in a single polypeptide chain, providing an example of a bifunctional enzyme. It catalyzes two successive reactions that transfer branches.

C. Phosphoglucomutase

Phosphoglucomutase catalyzes the reversible reaction involving the interconversion of the end product of the glycogen phosphorylase reaction, G1P, into G6P either for entry into glycolysis in muscle or hydrolysis to glucose in liver. The catalytic site of an active phosphoglucomutase molecule contains a phosphorylated serine residue. In step 1, the phosphoryl group is transferred from the Ser residue to the C-6 hydroxyl group of G1P to form glucose-1,6-bisphosphate (GBP) intermediate. In step 2, the phosphoryl group at C-1 of GBP is shuttled back to the same serine residue producing glucose 6-

6 | P a g e phosphate and regenerating the phosphoenzyme. The role of GBP in the interconversion of the phosphoglucoses is like that of 2,3-bisphosphoglycerate (2,3-BPG). Therefore, the presence of small amounts of GBP is necessary to keep phosphoglucomutase fully active.

There are three major pathways of glucose utilization, fates of glucose, in animals and vascular plants. Glucose may be stored as storage polysaccharide such as glycogen and starch or as sucrose; oxidized to generate a three-carbon compound (pyruvate) via glycolysis to provide ATP and metabolic intermediates; or oxidized via the pentose phosphate (phosphogluconate) pathway to yield ribose 5- phosphate for nucleic acid synthesis and NADPH for reductive biosynthetic processes. The glucose 6- phosphate formed from glycogen can enter glycolysis in skeletal muscle and serve as an energy source to support muscle contraction. Glycolysis is the pathway with the largest flux of carbon in most cells. In contrast to muscles, glucose is not a major fuel for the liver. Although not the only possible fates for glucose, these three pathways are the most significant in terms of the amount of glucose that flows through them in most cells.

D. Glucose 6-phosphatase

Phosphorylated glucose produced by glycogen breakdown cannot be transported out of cells. The liver contains a hydrolytic enzyme, glucose-6-phosphatase, which cleaves the phosphoryl group of G6P to form free glucose and orthophosphate. Glucose 6-phosphatase is an integral membrane protein of the smooth endoplasmic reticulum. It is predicted to contain nine transmembrane helices with its catalytic site facing the lumen of the ER. Glucose 6-phosphate formed in the cytosol is transported into the ER lumen by a specific G6P translocase (T1) and hydrolyzed at the luminal surface by the glucose 6- phosphatase. The resulting Pi and glucose are shuttled back into the cytosol by two different transporters T2 and T3, respectively. This hydrolytic enzyme enables glucose to leave the hepatocyte via GLUT2 glucose transporter in the plasma membrane.

The conversion of glycogen into free glucose takes place mainly in the liver and kidney but not in other tissues. G6P derived from the breakdown of glycogen in a hepatocyte has three possible fates: passage into glycolysis for the production of ATP, passage into the pentose phosphate pathway for the production of NADPH and pentose phosphates, or hydrolysis to free glucose and phosphate to be released into the bloodstream. A major function of the liver is to maintain a nearly constant level of glucose in the blood. Glucose released by the liver is taken up primarily by the brain, skeletal muscle,

7 | P a g e and red blood cells. Glucose 6-phosphatase is absent from most tissues including muscle and adipose tissue. Consequently, these tissues do not contribute glucose to the blood.

Glycogen synthesis

Glycogen synthesis or glycogenesis is the anabolic pathways from glucose to glycogen. It requires an activated form of glucose in the form of uridine diphosphate glucose (UDPG). It takes place only place when glucose is abundant. The C-1 carbon atom of the glucosyl unit of UDP-glucose is activated because its hydroxyl group is esterified to the diphosphate moiety of UDP. This “high-energy” nucleotide sugar is a glucose donor for polymerization reactions. Enzymes involved in glycogen synthesis pathway are UDP– glucose pyrophosphorylase, glycogenin, glycogen synthase, and glycogen branching enzyme. UDPG pyrophosphorylase catalyzes the formation of UDPG from UTP and G1P. New glycogen particles begin with the autocatalytic formation of a glycosidic bond between the glucose of UDP-glucose and a Tyr residue glycogenin. Glycogen synthase cannot catalyze de novo polymerization of glycogen. Instead, it requires a primer, usually a preformed linear or branched chain of at least eight glucose residues. Glycogenin catalyzes the assembly of the primer, glycogen synthase elongates the primer, and glycogen branching enzyme catalyzes the formation of branches.

A. UDP–glucose pyrophosphorylase

The role of nucleotide sugars in biochemistry was discovered by the Argentine biochemist Luis Leloir in 1957. UDP-glucose is the immediate donor of glucose residues in the biosynthesis of glycogen. UDP– glucose pyrophosphorylase catalyzes the synthesis of UDP-glucose from glucose 1-phosphate and uridine triphosphate (UTP). The reaction is a phosphoanhydride exchange in which the phosphoryl oxygen of G1P attacks the α-phosphorus atom of UTP to form UDPG and release the outer two phosphoryl residues of UTP as pyrophosphate (PPi). The PPi formed is rapidly hydrolyzed in an exergonic reaction by the omnipresent enzyme inorganic pyrophosphatase. UDP is phosphorylated back into UTP in a reaction catalyzed by nucleoside diphosphokinase employing ATP.

B. Glycogenin

Glycogenin primes the initial sugar residues for the synthesis of glycogen by glycogen synthase through its Mn2+ requiring intrinsic glucosyltransferase activity. The very first step in the synthesis of a new glycogen molecule is the self-catalyzed transfer of a glucose residue from UDP-glucose to the hydroxyl group of Tyr-194 of glycogenin. This is followed by sequential addition of seven more glucose residues to

8 | P a g e form a primer for the initiation of glycogen synthesis. The primer can be acted upon by glycogen synthase. Glycogenin, the primer polymerizing enzyme, is composed of two identical subunits. UDP- glucose is bound to a Rossman fold near the amino terminal portion of the enzyme. Rossmann-like fold is common to the nucleotide-binding domains of most glycosyltransferases. Each subunit catalyzes the formation of α-1,4-linked oligosaccharide units. Consequently, each glycogen molecule is associated with one molecule of glycogenin covalently attached to the single reducing end of the glycogen molecule and one molecule of glycogen synthase buried within the particle. Humans have glycogenin (muscle form) and a second isoform in liver glycogenin-2.

Glycogenin catalyzes two distinct types of reactions. The first reaction type involves initial attack by the hydroxyl group of Tyr 194 on C-1 of the glucosyl moiety of UDP-glucose to form glucosylated Tyr residue. The glycosidic bond in the product has the same configuration about the C-1 of glucose as the substrate UDPG, suggesting that the first reaction type takes place in two steps. Step one is a nucleophilic attack by Asp 162, forming a temporary intermediate with inverted configuration. Step two is a second nucleophilic attack by Tyr 194 to restore the starting configuration. The second reaction type involves attack by the C-4 hydroxyl group of the terminal glucose on the C-1 of another UDP-glucose molecule. Multiple repeats of the second reaction type forms a nascent glycogen molecule of eight glucose residues attached by α(1→4) glycosidic linkages. In both reaction types, the UDPG substrate is bound to a Mn2+ ion that is essential to catalysis through its phosphates. Mn2+ functions as an electron-pair acceptor (Lewis acid) to stabilize the UDP leaving group.

C. Glycogen synthase

Glycogen synthase is a key regulatory enzyme in glycogen synthesis which catalyzes the transfer of the glucosyl unit of UDPG to the C-4 OH group on one of the non-reducing terminal ends of a growing chain to form an α(1→4)-glycosidic bond. However, it cannot simply link together two glucose residues; it can only extend an already existing glucan chain of 7 to 8 glucose residues initiated by glycogenin. Thus, every glycogen molecule has a glycogenin molecule at its core in the cytoplasm. Mammals including humans have two isozymes of glycogen synthase: one is specific to the liver while the other is expressed in muscle and other tissues. The two isoforms are nearly 70% identical in sequence.

One proposed mechanism for the glycogen synthase reaction involves a glucosyl oxonium ion intermediate like those of glycogen phosphorylase and lysozyme. Glycogen synthase can be inhibited by 1,5-gluconolactone, a transition state analog that mimics the oxonium ion’s half-chair geometry.

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Glycogen synthase is a member of the large glycosyltransferase family. Bacteria and plants express starch/glycogen synthases which employ ADP–glucose as glucose donors. They exhibit very little sequence similarity to animal glycogen synthases. On the other hand, despite no detectable sequence similarity, structural studies have revealed that glycogen synthase is homologous to glycogen phosphorylase. The binding site for UDP-glucose in glycogen synthase corresponds in position to the pyridoxal phosphate in glycogen phosphorylase.

D. Glycogen branching enzyme

Glycogen is a branched polymer of glucose. However, glycogen synthase catalyzes the formation of only α(1→4)-glycosidic bonds to yield a linear chain. Branching points must be introduced by the amylo- (1→4) to (1→6) transglycosylase or glycosyl-(4→6)-transferase, also called glycogen-branching enzyme. Glycogen branching enzyme catalyzes the transfer of a terminal block of α(1→ 4)-linked 6 or 7 glucosyl residues from the nonreducing end of a glycogen chain having at least 11 residues to the C-6 OH group of a glucose residue at a more interior site of the same or another glycogen chain. The new branch point must be at least 4 residues away from a preexisting one. Hence, branching involves the breaking of an old α-1,4 link and the formation of a new α-1,6 link. A branch is created by an α-1,6-glycosidic bond at about every twelfth residue.

Significance of branching

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The physiological significance of the highly branched structure of glycogen is maximizing the density of stored glucose units consistent with the need to rapidly mobilize it under conditions of high metabolic demand. Branching increases the solubility of glycogen and the number of nonreducing ends. This presents a large number of terminal residues at the surface of the glycogen granule, accessible to glycogen metabolizing enzymes. Branching permits rapid degradation of glycogen through the simultaneous release of the glucose units at the end of every branch. Thus, branching increases the rate of glycogen synthesis and degradation. Three related parameters must be optimized to facilitate glycogen mobilization: the number of tiers of branches in a glycogen molecule, the number of branches per tier, and the average chain length per tier. Typically, a glycogen granule contains 12 tiers of branches, with 2 branches per tier and branch lengths averaging 13 residues. Mathematical analysis suggests that these values are close to optimal for mobilizing the greatest amount of glucose in the shortest possible time.

Thermodynamics of glycogen metabolism

The phosphorylase reaction is at equilibrium at 25°C when the ratio of [Pi] to [G1P] is 3.5. Consequently, this reaction is readily reversible in vitro. However, the reaction proceeds far in the direction of glycogen breakdown in vivo because the concentration ratio is usually greater than 100 making glycogen breakdown exergonic. The phosphorolytic cleavage of glycogen is energetically advantageous because the released sugar is already phosphorylated. Hydrolytic cleavage would yield glucose which needs to be phosphorylated at the expense of a molecule of ATP. Conversely, glycogen synthesis from G1P under physiological conditions is thermodynamically unfavorable without free energy input. Since the direct conversion of G1P to glycogen and Pi is thermodynamically unfavorable under all physiological concentration ratios, glycogen biosynthesis requires an additional exergonic step utilizing sugar nucleotides.

Biosynthetic and degradation pathways rarely operate by precisely the same reactions in the forward and reverse directions. Glycogen metabolism provided the first known example of this important principle. Only one molecule of ATP is hydrolyzed to incorporate glucose 6-phosphate into glycogen. For each molecule of G1P that is converted to glycogen and then regenerated, one molecule of UTP is hydrolyzed to UDP and Pi. Therefore, the cyclic synthesis and breakdown of glycogen is an “engine” powered by UTP hydrolysis. Glycogen is an efficient storage form of glucose with overall efficiency of storage nearly 97%. Besides, muscle cells have no transporters for glucose-1-phosphate, which is negatively charged under physiological conditions. Hence it cannot diffuse out of the cell and essentially

11 | P a g e sequestered for utilization in muscle cells. Glycogen metabolism is exquisitely regulated by the independent control of its anabolic and catabolic pathways

Control of glycogen metabolism

Glycogen metabolism is coordinately controlled by mechanisms that regulate key individual regulatory enzymes. To this end, glycogen phosphorylase (GP) and glycogen synthase (GS) are under stringent control such that glycogen is either synthesized or utilized according to cellular needs. Hence, glycogen synthesis and breakdown are reciprocally controlled. An important control mechanism prevents glycogen from being synthesized at the same time as it is being broken down. Both glycogen synthase and glycogen phosphorylase are subjected to direct allosteric control and enzyme-catalyzed covalent modifications. The rates of both glycogen synthesis and degradation are under allosteric control by effectors such as ATP, G6P, glucose and AMP. Glycogen phosphorylase is stimulated and glycogen synthase is inhibited when there is high demand for ATP signaled by low [ATP], low [G6P], low [glucose], and high [AMP]). Covalent modifications of GP and GS alter the structures of these enzymes so as to change their responses to allosteric regulators. Covalent modification systems can respond to a greater number of allosteric stimuli and exhibit greater flexibility in their control patterns. The covalent modification reactions in turn are themselves ultimately under hormonal control through enzymatic cascades.

Covalent modification of enzymes by cyclic cascades

By convention, the more active form of an interconvertible target enzyme has the suffix -a- and the less active form has the suffix -b- whereas, the modified form of the enzyme bears the prefix -m- and the original or unmodified form bears the prefix -o-. A general scheme for a monocyclic enzyme cascade involves one target (E), one modifying (F), one demodifying enzymes (R) and two effectors (e1 and e2). The target enzyme (E) can more active in the modified form (m-E-a) and less active in the unmodified form (o-E-b). The F and R enzymes are allosterically converted from their inactive to their active conformations on binding their respective effectors, e1 and e2. In contrast, a bicyclic enzyme cascade involves covalent modification of the metabolic target enzyme (E) as well as one of the modifying enzymes (F). The interconversion of both E and F requires distinct, enzyme-catalyzed covalent modification and demodification reactions. The activities of both glycogen phosphorylase and glycogen synthase are controlled by bicyclic cascades in which the primary intracellular signal, e1, is adenosine- 3’,5’-cyclic monophosphate (cAMP).

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Glycogen phosphorylase

Glycogen phosphorylase is one of the first allosteric enzymes identified and one of the most studied regulatory enzymes. It is regulated by several allosteric effectors that signal the energy state of the cell as well as by reversible phosphorylation, which is responsive to hormones such as insulin, epinephrine, and glucagon. Glycogen phosphorylase is a dimer that exists in two interconvertible forms: an inactive form (phosphorylase-b) that requires AMP for activity and an active form (phosphorylase-a) that is active without AMP. Each of these two forms exists in equilibrium between an active relaxed (R) state and a much less active tensed (T) state. The equilibrium for phosphorylase b favors the less-active T state, whereas the equilibrium for phosphorylase a favors the active R state. The structural differences between the active (R) and inactive (T) conformations of glycogen phosphorylase can be understood in terms of the symmetry model of allosteric regulation. The T-state enzyme has a buried active site and hence a low affinity for its substrates, whereas the R-state enzyme has an accessible catalytic site and a high-affinity phosphate binding site.

Allosteric regulation of glycogen phosphorylase

AMP binds to the R state of the enzyme at its allosteric effector site and promotes the T→R conformational shift. This is accompanied by the movement of the phosphoryl group of Ser-14 from the surface of the T-state to a position buried a few angstroms beneath the protein’s surface at the dimer interface in the R-state state. AMP’s adenine, ribose, and phosphate groups bind to separate segments of the polypeptide chain triggering a concerted T→R transition and tertiary movements. The enzyme’s 2- fold symmetry is thereby preserved in accordance with the symmetry model of allosteric regulation. ATP binds to the allosteric effector site, but in the T state, so that it inhibits rather than promotes the T→R

13 | P a g e conformational shift. ATP competes with AMP for binding to phosphorylase and inhibits phosphorylase activity.

The liver form

The role of glycogen breakdown in the liver is to liberate glucose for export to other tissues, such as skeletal muscle and the brain, when low blood-glucose level is signaled by glucagon. Consequently, phosphorylase-a form is the default state of liver phosphorylase: glucose is generated from glycogen degradation unless the enzyme is signaled otherwise. It is largely unresponsive to AMP and is mostly in the R state unless there is a high level of glucose. Liver phosphorylase is regulated by the blood glucose level. The phosphorylase-a form in the liver is inhibited by glucose which shifts the equilibrium to the T state. Thus, glycogen is not mobilized when glucose is already abundant. Unlike the enzyme in muscle, the liver phosphorylase is insensitive to regulation by AMP because the liver does not undergo the dramatic changes in energy charge seen in a contracting muscle. Hence liver glycogen phosphorylase is inhibited by glucose, G6P and ATP and but not AMP.

The muscle form

The physiology of skeletal muscle differs from that of the liver in three fundamental ways. Muscle undergoes very large changes in its demand for ATP which is supported by glycolysis as it goes from rest to vigorous contraction. Muscle uses its stored glycogen only for its own needs and it lacks the enzymatic machinery for gluconeogenesis. Consequently, phosphorylase-b form is the default state of muscle phosphorylase: glucose is not generated from glycogen degradation during muscle rest. Glucose degradation is signaled by muscle contraction. Muscle phosphorylase is regulated by the intracellular energy charge. In resting muscle, the concentrations of ATP and G6P are high enough to inhibit phosphorylase b which is mostly in the T state. In vigorously contracting muscle, AMP accumulates to high concentration as a result of ATP breakdown. AMP binds to a nucleotide-binding site and stabilizes the conformation of phosphorylase b in the active R state, speeding the release of G1P from glycogen. Hence, muscle glycogen phosphorylase is activated by AMP and inhibited by ATP and G6P.

Unlike hepatocytes, myocytes lack receptors for glucagon but contain a reserve of GLUT4 sequestered in intracellular vesicles. The muscle isozyme of pyruvate kinase is not phosphorylated by PKA, so glycolysis is not turned off when [cAMP] is high. In fact, cAMP increases the rate of glycolysis in muscle, probably by activating glycogen phosphorylase. Skeletal muscles consist of both slow-twitch (Type I) and fast- twitch (Type II) fibers. Muscles designed to contract slowly and steadily are enriched in slow-twitch (type

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I) fibers. Type I muscle fibers, which power endurance activities, rely predominantly on oxidative phosphorylation to derive energy. These fibers are powered by fatty acid degradation and are rich in mitochondria. The flight muscles of migratory birds such as ducks and geese, which need a continuous energy supply, are rich in slow-twitch fibers and therefore such birds have dark brown breast meat characteristic color of heme-containing cytochromes.

In contrast, fast-twitch fibers, which predominate in muscles capable of short bursts of rapid activity, are nearly devoid of mitochondria, so that they must obtain nearly all of their ATP through anaerobic glycolysis, for which they have a particularly large capacity. The flight muscles of game birds, such as chickens and turkeys, which are used only for short bursts (often to escape danger), consist mainly of fast-twitch fibers that form pale colored meat. Type IIa muscle fibers are “trainable”; that is, endurance training enhances their oxidative capacity. Type IIb muscle fibers, which power burst activities such as sprinting and weight lifting, use glycogen as their main fuel. In type IIb muscle fibers, the transition of phosphorylase b between the active R state and the less-active T state is controlled by the energy charge of the muscle cell. ATP acts as a negative allosteric effector by competing with AMP. G6P also binds at the same site as ATP and stabilizes the less-active state of phosphorylase b by feedback inhibition.

A bicyclic cascade of glycogen phosphorylase

The bicyclic cascade for glycogen phosphorylase comprises glycogen phosphorylase, the target enzyme of the cascade, and the actions of three enzymes: phosphorylase kinase (PhK), protein kinase A (PKA), and phosphoprotein phosphatase-1 (PP1). In both liver and muscle, phosphorylase b is converted into phosphorylase a by phosphorylation of a Ser-14 in each subunit by the regulatory enzyme PhK. Phosphorylation promotes the conversion of phosphorylase b to phosphorylase a. PKA phosphorylates and activates PhK whereas PPA1 dephosphorylates and deactivates both glycogen phosphorylase-a and PhK. In this cascade, o-phosphorylase b (unmodified, inactive) is the form under allosteric control by AMP, ATP, and G6P. Phosphorylation shifts the enzyme’s T → R equilibrium in favor of the R state and yields m-phosphorylase-a (modified, active). Phosphorylation also removes the effects of allosteric modulators. Glycogen phosphorylase can also be regulated by acetylation. Acetylation not only inhibits the enzyme, but also enhances dephosphorylation by promoting the interaction with PP1.

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Glycogen synthase

Glycogen synthesis takes place in virtually all animal tissues but it is especially prominent in the liver and skeletal muscles. The key regulatory enzyme in glycogen synthesis is glycogen synthase. It exists in two interconvertible forms: an active unmodified form (o-glycogen synthase-a) and a phosphorylated physiologically inactive b form (m-glycogen synthase-b). The key means of regulating glycogen synthase is by allosteric regulation of the phosphorylated form of the enzyme. The modified form is strongly activated by G6P and strongly inhibited by physiological concentrations of ATP, ADP, and Pi. G6P is a powerful activator of the enzyme, stabilizing the R state of the enzyme relative to the T state. It binds to an allosteric site on m-glycogen synthase-b and induces a conformational change that exposes the phosphoryl groups the synthase to the surface of the protein, thereby making them better substrates for dephosphorylation by phosphoprotein phosphatase-1. This form of glycogen synthase can be regarded as a G6P sensor. In contrast, the activity of the unmodified enzyme is essentially independent of allosteric effectors.

Bicyclic cascade of glycogen synthase

The interconversion of the two forms glycogen synthase is regulated by covalent modification through phosphorylation and dephosphorylation cycles. However, covalent modification of glycogen synthase plays more of a fine-tuning role. The phosphorylated form of glycogen synthase (m-GS-b) is inactive unless its allosteric activator, G6P, is present. The bicyclic cascade for glycogen synthase comprises

16 | P a g e glycogen synthase, the target enzyme of the cascade, and the actions of several enzymes including glycogen synthase kinase (GSK), phosphorylase kinase (PhK), casein kinase II (CKII), and phosphoprotein phosphatase-1 (PP1). In both liver and muscle, glycogen synthase-a form is converted into glycogen synthase-b form by phosphorylation of the hydroxyl side chains of several Ser residues on both subunits. Glycogen synthase is remarkable for its ability to be phosphorylated at multiple sites by several protein kinases including GSK (which is under the control of insulin), CaMKI (which is activated by the presence of Ca2+), PhK, PKA and protein kinase C (PKC).

The most important regulatory kinase for glycogen synthase is glycogen synthase kinase 3 (GSK3). The action of GSK3 is hierarchical. Firstly, GSK3 associates with glycogen synthase. This association creates a new phosphorylation site on glycogen synthase for another protein kinase, an event called priming. Secondly, casein kinase II (CKII) phosphorylates the glycogen synthase on a nearby residue. Thirdly, GSK3 adds phosphoryl groups to three Ser residues near the carboxyl terminus of glycogen synthase, strongly inactivating it. GSK3 action requires prior phosphorylation (priming) by CKII. GSK3 converts glycogen synthase to the inactive form (m-GS-b). Other GSK3 targets include cytoskeletal proteins and proteins essential for mRNA and protein synthesis. These targets, like glycogen synthase, must first undergo a priming phosphorylation by another protein kinase before they can be phosphorylated by GSK3.

Phosphorylation has opposite effects on the enzymatic activities of glycogen synthase and glycogen phosphorylase. In muscle, epinephrine activates PKA, which phosphorylates the glycogen-targeting protein GM on a site that causes dissociation of PP1 from glycogen. PKA or PKB phosphorylates a Ser residue near the amino terminus of GSK3. This phosphorylation converts a region of GSK3 into a “pseudosubstrate” that folds into the site at which the priming phosphorylated Ser residue normally binds. Inhibitory phosphorylation prevents GSK3 from binding the priming site of a real substrate, thereby inactivating GSK3. PP1 removes the phosphoryl groups from the pseudosubstrate region of GSK3 and activates it tipping the balance in favor of phosphorylation of glycogen synthase by GSK3. Hence, epinephrine inhibits glycogen synthesis in muscles.

In liver, the conversion of m-GS-b to o-GS-a is promoted by PP1 which is bound to the glycogen particle. PP1 removes the phosphoryl groups from the three Ser residues phosphorylated by GSK3. PP1 in liver is directly inhibited by active phosphorylase a, preventing it from activating glycogen synthase. Glucose also promotes dephosphorylation of glycogen synthase indirectly by relieving inhibition of PP1 by phosphorylated glycogen phosphorylase. Glucose binding to glycogen phosphorylase-a form induces

17 | P a g e conformational change that favors dephosphorylation to glycogen phosphorylase b. The b-form cannot inhibit PP1.

Metabolic control analysis has been applied to glycogen metabolism. Investigators have used nuclear magnetic resonance (NMR) as a noninvasive means to determine the concentration of glycogen and metabolites in the five-step pathway from glucose in the blood to glycogen in myocytes. They found that the flux control coefficient for glycogen synthase was smaller than that for the steps catalyzed by the glucose transporter and hexokinase. This finding contradicts the conventional wisdom that glycogen synthase is the locus of flux control. Metabolic control analysis suggests that the importance of the phosphorylation/dephosphorylation of glycogen synthase is related instead to the maintenance of metabolite homeostasis—that is, regulation, not control.

Integration of glycogen metabolism

The activities of key regulatory enzymes involved in glycogen metabolism are adjusted to meet the needs of the cell by the action of allosteric effectors. Superimposed on the action allosteric control mechanisms are regulation by phosphorylation. Protein kinases play key roles in the signaling pathways by which many hormones, growth factors, neurotransmitters, and toxins affect the functions of their target cells and control metabolic pathways. Many protein kinases are themselves phosphoproteins whose activities are controlled by phosphorylation, often at their activation loops. The bicyclic cascades, one controlling the rate of glycogen breakdown and the other controlling the rate of glycogen synthesis, are intimately related by the actions of hormones, secondary messengers, and protein kinases such as PKA, PhK, and PP1. Hormones are important coordinators of the control of carbohydrate metabolism to meet the needs of the entire organism.

The metabolism of fats and fatty acids is very closely integrated to that of carbohydrates by hormonal signals. Hormonal signals, changes in diet and exercise are all important in integrating fat metabolism with that of carbohydrates. Carbohydrate metabolism is largely regulated by the two peptide hormones, insulin and glucagon, acting in opposition and two adrenal hormones, epinephrine/adrenaline and norepinephrine/noradrenaline. Carbohydrate metabolisms in the well-fed state, during fasting, and in the fight-or-flight response are signaled by insulin, glucagon, and epinephrine, respectively. Hormonal stimulation of cells at their plasma membranes occurs through the mediation of transmembrane proteins called receptors. Some hormones act at cell surfaces through G-protein coupled receptors (GPCR). Both muscle and liver cells have abundant insulin and adrenergic receptors (receptors

18 | P a g e responsive to epinephrine and norepinephrine), whereas glucagon receptors are more abundant in liver cells than in skeletal muscles.

Activated G proteins transmit the signal from the activated GPCR receptor to adenylyl cyclase (AC; also called adenylyl cyclase). Stimulated AC releases second messengers inside the cell. Second messengers such as cAMP and Ca2+ are intracellular mediators of the externally received hormonal signals. The intracellular [cAMP] is a function of the ratio of its rate of synthesis from ATP by activated AC and its rate of breakdown to AMP by cAMP-phosphodiesterases (cAMP-PDEs). The elevated cytoplasmic level of cAMP activates PKA which sets off a cascade of phosphorylations. PKA activates phosphorylase b kinase (PhK), which in turn activates glycogen phosphorylase and inactivate glycogen synthase through phosphorylation. On the other hand, another secondary messenger, Ca2+, is released from stores in the endoplasmic reticulum. Binding of Ca2+ to calmodulin leads to a partial activation of phosphorylase kinase. The same glucagon- and epinephrine-triggered cAMP cascades that initiate glycogen breakdown also shut off glycogen synthesis.

Protein kinase A (PKA)

Protein kinase A (PKA), also called cAMP-dependent protein kinase (cAPK), is a crucial regulatory enzyme that phosphorylates specific Ser and/or Thr residues of numerous cellular proteins, including phosphorylase kinase and glycogen synthase. PKA is activated by epinephrine or glucagon through a series of steps that ultimately increase the intracellular concentration of cAMP. All PKA substrate proteins contain PKA’s consensus recognition sequence, Arg-Arg-X-Ser/Thr-Y, where Ser/Thr is the phosphorylation site, X is any small residue, and Y is a large hydrophobic residue. PhK is responsible for activating phosphorylase by transferring a phosphoryl group to its Ser residue to initiate glycogen breakdown. Glycogen synthase kinase adds phosphoryl groups to glycogen synthase, but this phosphorylation leads to a decrease in enzymatic activity.

The [cAMP] is absolutely required for the activity of PKA since it determines the fraction of PKA in its active form and thus the rate at which it phosphorylates its substrates. In the absence of cAMP, PKA is an inactive heterotetramer consisting of two regulatory (R) and two catalytic (C) subunits, R2C2, in which the R subunit competitively inhibits its C subunit. The N-terminal portion of the R subunit has a peptide segment, also known as autoinhibitor segment or a pseudo–target sequence. The pseudo-target sequence, which resembles the C subunit’s substrate peptide, occupies in the C subunit’s active site

19 | P a g e cleft. The binding of cAMP to the inhibitory regulatory subunits triggers their dissociation from the catalytic subunits. The catalytically active monomeric subunits become free.

Phosphorylase kinase (PhK)

Phosphorylase kinase phosphorylates phosphorylase b and promotes its conversion into the active phosphorylase-a form. PhK is an autoinhibited kinase. Autoinhibited kinases have either N- or C-terminal “pseudosubstrate” sequence that binds at or near the enzyme’s active site. The subunit composition of

PhK in skeletal muscle is (αβγδ) 4. It contains two (αγδ) 2 lobes that are joined by β4-bridge. The isolated γ subunit is the catalytic subunit with full catalytic activity whereas the remaining α, β, and δ subunits are regulatory with inhibitory roles. The δ subunit, also known as calmodulin (CaM), is calcium binding protein that confers Ca2+ sensitivity to the complex. CaM is a calcium sensor which functions as a Ca2+- activated switch with four Ca2+-binding sites. The catalytic subunit consists of an N-terminal kinase domain which carries the active site and a C-terminal regulatory domain which contains a CaM-binding peptide and an overlapping autoinhibitor segment. The N-terminal catalytic site maintains an active conformation but it is inactivated by its C-terminal autoinhibitor segment in the absence of Ca2+.

CaM is a ubiquitous eukaryotic Ca2+-binding regulatory protein that undergoes an extensive conformational change upon calcium biniding. It participates in numerous cellular regulatory processes and stimulates many enzymes in eukaryotes including phosphorylase kinase in response to Ca2+ binding. CaM has a curious dumbbell-like shape containing two globular domains connected by a seven-turn α- helix. Each of the globular domains, both of which are formed by nearly superimposable helix–loop– helix motifs known as EF hands, has two high-affinity Ca2+-binding sites. The Ca2+ ion is octahedrally coordinated by oxygen atoms from the backbone and side chains of the loop as well as from a protein- associated water molecule. The central α-helix serves as a flexible linker rather than as a rigid spacer. The Ca2+ binding segment on CaM subunit of PhK is in close proximity with the autoinhibitory segment of the catalytic subunit. When Ca2+ binds to phosphorylase b kinase through its CAM subunit, the accompanying conformational change extracts the autoinhibitory sequence from the enzyme’s active site, thereby activating the enzyme.

EF hands constitute the Ca2+-binding sites in many proteins that function to sense the level of Ca2+. Calcium binding to CaM induces extensive conformational change that exposes an otherwise buried Met-rich hydrophobic patch. Many Ca2+-regulated proteins including the γ subunit of PhK have CaM- binding domains that recognize the exposed peptide segment on activated CaM with high affinity. The

20 | P a g e high affinity binding of the CaM peptide segment to CaM-binding domain extracts the autoinhibitor from the active site of CaM-binding proteins activating this autoinhibited enzyme. This is called intrasteric mechanism. The physiological significance of Ca2+ activation of PhK is to couple muscle contraction and glycolysis. Nerve impulses trigger muscle contraction through the release of Ca2+ from intracellular reservoirs in sarcoplasmic reticulum. The resulting transient increase in cytosolic [Ca2+] induces glycogen breakdown that supplies glycolysis, which in turn, generates the ATP required for muscle contraction.

The stimulation of PhK is one step in a signal-transduction cascade initiated by signal molecules such as glucagon and epinephrine. PhK is activated by both allosteric regulation via calcium ions and covalent modification via phosphorylation. There are two preconditions for PhK to be fully active: Ca2+ must be present and the protein must be phosphorylated. PKA phosphorylates α and β subunits of PhK. The β subunit is phosphorylated first, followed by the phosphorylation of α subunit. Phosphorylation and Ca2+ has synergistic effect on the activity of PhK. Phosphorylation helps to activate PhK at much lower Ca2+ concentrations than otherwise. Maximal enzyme activity is achieved with the phosphorylation of both the β and α subunits of PhK in the presence of Ca2+. The release of Ca2+ induces both muscle contraction and glycolysis.

Signal amplification

The enzymes that modify and demodify a metabolic target enzyme can themselves be under allosteric control by secondary messengers. Enzyme cascade is a chain of catalysis in which a catalyst activates a catalyst, which in turn activates another catalyst via secondary messengers such as cAMP. Enzyme cascades have enormous signal amplification potential in their responses to variations in effector concentrations. Because of the amplifying properties of the cyclic cascades, the binding of a small number of hormone molecules to cell-surface receptors leads to the release of a very large number of secondary messengers. A small change in [cAMP] results in a large change in the fraction of enzymes in their phosphorylated forms.It is therefore possible for a small change in concentration of an allosteric effector of a modifying enzyme to cause a large change in the concentration of an active, modified target enzyme and ultimately a large number of metabolic products.

Signal termination

Target cells can revert quickly to a resting state due to the action of specific mechanisms of termination of signal transduction or attenuation and reuptake of the signaling molecule. Glycogen breakdown must

21 | P a g e be rapidly terminated when necessary. It is crucial that the high-gain system of glycogen breakdown be switched off quickly to prevent the wasteful depletion of glycogen after energy needs have been met. The inherent GTPase activity of the G protein converts the bound GTP into inactive GDP, and phosphodiesterases always present in the cell convert cAMP into AMP. Both phosphorylase kinase and glycogen phosphorylase are inactivated when dephosphorylated by phosphoprotein phosphatase 1 (PP1). PP1 accomplishes the task of shutting down phosphorylated proteins that stimulate glycogen breakdown. Simultaneously, it activates glycogen synthesis.

Phosphoprotein phosphatase-1 (PP1)

The steady-state phosphorylation levels of most enzymes involved in bicyclic cascades are maintained by the opposing actions of protein kinase-catalyzed phosphorylations and the hydrolytic dephosphorylations catalyzed by phosphoprotein phosphatases. Phosphoprotein phosphatase-1 (PP1) catalyzes the hydrolysis and removal of phosphoryl groups from phosphorylated serine and threonine residues in several phosphoproteins including both α and β subunits of phosphorylase kinase, m- glycogen phosphorylase a, and m-glycogen synthase-b. PP1 plays a central role in regulating glycogen metabolism. It decreases the rate of glycogen breakdown by reversing the regulatory effects of the phosphorylation cascade. Moreover, PP1 removes phosphoryl groups from m-glycogen synthase-b to accelerate the rate of glycogen synthesis. The binding of glucose to m-phosphorylase-a shifts its allosteric equilibrium from the active R form to the inactive T form. This conformational change renders the phosphoryl group on Ser-14 a substrate for PP1. PP1 binds tightly to m-phosphorylase-a in the T state but it is inactive when bound to m-phosphorylase-a in the R state.

The catalytic subunit PP1, designated as PP1c, is a 37-kDa single-domain protein which hydrolyzes phosphoryl groups on Ser/Thr residues via a single step mechanism. It contains a binuclear metal ion center (such as Mn2+) which activates a water molecule (promotes its ionization to OH-) for nucleophilic attack on the phosphoryl group. PP1c does not exist free in the cytosol, but it is tightly bound to its target proteins via one of the intermediary regulatory subunits. Glycogen-targeting proteins are a family of regulatory proteins with masses of approximately 120 kDa which have modular structures and domains that participate in interactions with glycogen, with the catalytic subunit, and with target enzymes. They act as scaffolds, bringing together the phosphatase and its substrates on the glycogen particle. Therefore, glycogen-targeting proteins bind other proteins such as glycogen phosphorylase, phosphorylase kinase, glycogen synthase, and phosphoprotein phosphatase-1 to glycogen particles. In

22 | P a g e skeletal muscle and heart, the most prevalent glycogen-targeting protein is GM, whereas in the liver, the most prevalent regulatory subunit is GL.

The antagonistic effects of insulin and epinephrine on glycogen metabolism in muscle occur through their effects on the PP1 catalytic subunit, PP1c, via its glycogen-bound GM subunit. In response to insulin and epinephrine, the activity of PP1c and its affinity for the GM subunit are regulated by phosphorylation of the GM subunit at two different sites. Phosphorylation of site 1 by insulin-stimulated protein kinase activates PP1c, whereas phosphorylation of site 2 by epinephrine-stimulated PKA inactivates PP1c. Site 2 is in the domain responsible for PP1c binding. Phosphorylation of GM at site 2 by PKA dissociates PP1c from the glycogen particle preventing its access to its substrates. In contrast to GM in muscle, GL in the liver is not subject to control via phosphorylation. The active form of phosphorylase and PP1c are localized to the glycogen particle by interactions with the GL subunit of PP1. The activity of PP1c and its affinity for the GL subunit are regulated allosteric binding of m-phosphorylase-a to GL that strongly inhibits the activity of PP1c. Glycogen synthase is also dephosphorylated by the mediation of GL.

PP1 is also inactivated by PKA and activated by allosteric effector G6P. PKA reduces the activity of PP1 by two mechanisms. It phosphorylates both the inhibitor of PP1 and the regulatory subunit (GM). All tissues contain inhibitor-1 which is a family of small proteins that, when phosphorylated, bind to PP1c and inhibit it. Phosphorylation of the inhibitor subunit by PKA inactivates the catalytic subunit PP1c and activates the dissociation of the phosphorylated regulatory subunit GM. Inhibitor-1 is phosphorylated and activated by PKA but dephosphorylated and inactivated by PP1c.

Insulin and the well-fed state

Normally immediately after a carbohydrate-rich meal has been digested, the blood glucose level rises. Eukaryotic cells have multiple glucose transporters. GLUT1 (for glucose transporter 1) is a glucose transporter in erythrocytes and many other tissues. The high-capacity glucose transporter, GLUT2, is particularly prominent in the intestine for absorption of dietary glucose. GLUT3 is expressed in neurons and the placenta whereas GLUT4 occurs mainly in muscle and fat cells. Liver and muscle, tissues that are highly active in glucose transport, contain only tiny amounts of GLUT1. However, liver cells and muscle cells contain ample quantities of GLUT2 and GLUT4 respectively. GLUT2 is also abundant in pancreatic β-cells which are freely permeable to glucose. GLUT2 catalyzes facilitated diffusion of glucose in both directions, at a rate high enough to ensure virtually instantaneous equilibration of glucose

23 | P a g e concentration in the blood and in the hepatocyte cytosol. High blood sugar decreases the release of glucagon from the pancreatic α-cells and increases the release of insulin from the pancreatic β-cells.

Binding of insulin to its receptor in the plasma membrane is the first step in the action of insulin. Insulin binding to its receptor activates a tyrosine protein kinase in the receptor. The activated receptor tyrosine kinase (RTK) phosphorylates a family of four homologous proteins named insulin-receptor substrate (IRS). Proteins such as IRS are also known as docking proteins because they function as platforms for the recruitment of a variety of downstream signaling molecules in response to the activation of their corresponding RTK. The IRS proteins all have an N-terminal “targeting” region that localizes the IRS to the inner surface of the plasma membrane, followed by a PTB domain that binds the IRS to a phosphotyrosine residue of an activated insulin receptor. The insulin receptor phosphorylates the IRS at one or more of its Tyr residues, converting them to SH2-binding sites. Apart from this, excess glucose is converted to G6P by hexokinase. Hexokinase in most cells is inhibited by its reaction product (G6P), has high glucose affinity, and obeys Michaelis–Menten kinetics with [glucose].

On the other hand, liver hexokinase IV (also called hexokinase D or glucokinase) is not inhibited by physiological concentrations of G6P, has a much lower affinity for glucose, and exhibits sigmoidal kinetics with a Hill constant rather than hyperbolic variation. Glucokinase (GK) regulates glucose homeostasis via an intracellular localization mechanism by glucokinase regulatory protein (GKRP). GK can freely shuttle between the nucleus and cytoplasm. However, GKRP is exclusively localized in the nucleus. At low glucose concentration, GK remains bound to GKRP in the nucleus, where it is unavailable to phosphorylate glucose. At elevated intracellular glucose concentration, GK dissociates from its nuclear regulator protein GKRP, enters the cytosol and phosphorylates glucose to G6P. GK forms G6P at a rate proportional to the concentration of glucose stimulating glycolysis and supplying the precursor for glycogen synthesis. G6P facilitates the dephosphorylation and activation of m-glycogen synthase-b to o- glycogen synthase a. Under these conditions, hepatocytes can store the excess glucose in the blood as glycogen.

When blood-glucose levels are high, insulin stimulates glycogen synthesis through two immediate effects: it triggers a cascade that leads to the phosphorylation and inactivation of glycogen synthase kinase 3 (GSK3) that prevents the phosphorylation and inactivation of glycogen synthase and it activates a protein phosphatase, perhaps PP1 in muscle, another phosphatase in liver. Hence, the two major insulin target enzymes appear to be GSK3 and PP1. The enzyme phosphoinositide 3-kinase (PI-3K) associates with phosphorylated IRS-1 through its SH2 domain. Activated PI-3K converts

24 | P a g e phosphatidylinositol 4,5-bisphosphate (PIP2) in the membrane to phosphatidylinositol 3,4,5- trisphosphate (PIP3). A protein kinase (PDK-1) is activated when bound to PIP3. Activated PDK-1 activates a second protein kinase B (PKB), which phosphorylates glycogen synthase kinase 3 (GSK3) in its pseudosubstrate region, inactivating it. Hence one way insulin triggers intracellular changes is by activating PKB that in turn phosphorylates and inactivates GSK3.

The inactivation of GSK3 allows protein phosphatase-1 (PP1) to dephosphorylate glycogen synthase, converting it to its active form. PP1 removes the phosphates from glycogen synthase, thereby activating the enzyme and allowing glycogen synthesis to restore glycogen reserves. Insulin also results in decreased [cAMP] and it triggers signal-transduction pathways that eventually lead to the activation of insulin-stimulated protein kinase that phosphorylates site 1 on the glycogen-targeting regulatory subunit of GM of PP1. This ultimately shift glycogen metabolism from glycogen breakdown to glycogen synthesis. Apart from this, acetylation of glycogen phosphorylase is stimulated by glucose and insulin, but inhibited by glucagon. In these four distinct ways involving GSK3, PP1, GM and phosphorylase, insulin stimulates glycogen synthesis immediately after feeding. Metabolic control analysis suggests that when the blood glucose level rises, insulin acts in muscle to increase glucose transport into cells by bringing GLUT4 to the plasma membrane, induce the synthesis of hexokinase, and activate glycogen synthase by covalent alteration. The first two effects of insulin increase glucose flux through the pathway (control), and the third serves to adapt the activity of glycogen synthase so that metabolite levels (glucose 6- phosphate, for example) will not change dramatically with the increased flux (regulation).

Maintenance of blood glucose level by the liver

Although insulin is the primary signal for glycogen synthesis, another is the concentration of glucose in the blood. Glycogen metabolism in the liver is important to maintain and buffer the blood concentration of glucose. The liver senses the concentration of glucose in the blood and phosphorylase a isoform in hepatocytes functions as a glucose sensor. High concentration of glucose shifts the T to R equilibrium of phosphorylase a toward the T state. Therefore, at high blood-glucose level, glucose inhibits phosphorylase a by binding only to the active site of the enzyme’s inactive T state. The R to T transition of muscle phosphorylase a is unaffected by glucose and is thus unaffected by the rise in blood-glucose levels. In its role as a glucose sensor, the glycogen phosphorylase of hepatocytes is essentially measuring the blood glucose level. This remarkable glucose-sensing system depends on three key elements: First, there is communication between the allosteric site for glucose and the serine phosphate. Second, PP1 is

25 | P a g e used simultaneously to inactivate phosphorylase and activate glycogen synthase. Third, the binding of the PP1 to phosphorylase a form prevents premature activation of glycogen synthase.

The allosteric site for glucose allows liver glycogen phosphorylase to function as its own glucose sensor and to respond appropriately to changes in blood glucose. Glucose binding to an allosteric site of the phosphorylase a isozyme of liver induces a conformational change that exposes its phosphorylated Ser residues to the action of PP1 which promotes dephosphorylation and inactivation of glycogen phosphorylase a. Glucose binding also favors the formation of the T state of phosphorylase which does not bind PP1, leading to the dissociation of PP1 from glycogen phosphorylase a form and glycogen particle. The free PP1 becomes active. It dephosphorylates glycogen m-phosphorylase-a and m-glycogen synthase-b, leading to sharp reduction of glycogen breakdown and the activation of glycogen synthesis. The conversion of R into T is accompanied by the release of PP1, which is then free to activate glycogen synthase. Consequently, the activity of glycogen synthase begins to increase only after most of phosphorylase a is converted into the T-form in hepatocyte cytosol in response to high blood glucose.

Glucose transport into cells

The tissue distributions of the various glucose transporters correlate with the response of these tissues to insulin. Glucose transport into hepatocytes is meditated by insulin-independent GLUT2, which is always present in the plasma membrane. Defective GLU2 in the liver result in symptoms resembling Type I glycogen storage disease. GLUT2 is also prominent in pancreatic β-cells which secrete insulin in response to increased [glucose] in the blood. The passive uptake of glucose by muscle and adipose tissues is mediated by the insulin-dependent glucose transporter GLUT4, which is mostly sequestered in membrane vesicles within the cell in the absence of insulin. The release of insulin in response to high blood glucose level triggers the movement of many GLUT4 molecules from vesicles to the plasma membrane where they allow increased glucose uptake. Insulin increases the rate of glucose transport across many cell membranes and hence intracellular glucose concentration by increasing the number of glucose transporters GLUT4 in the membrane. Consequently, liver is unresponsive to insulin whereas muscle and fat cells take up glucose when stimulated by insulin.

Gene expression

When blood-glucose levels are high, insulin inhibits glycogen breakdown and stimulates glycogen synthesis through delayed responses involving gene expression. Insulin regulates the expression of as many as 150 genes, including some related to fuel (carbohydrate and fat) metabolism. Insulin changes

26 | P a g e the expression of several genes involved in glycolysis and its regulation, lipid synthesis and its regulation, and enzymes involved in generation of the reducing power of the cell (NADPH) for fatty acid synthesis via the pentose phosphate pathway. Insulin slows down the expression of the genes for enzymes of gluconeogenesis while increasing the synthesis of glucose metabolizing proteins. Consequently, glucose becomes the fuel of choice (via glycolysis) for liver, adipose tissue, and muscle. Excess glucose is converted to glycogen and triacylglycerols for temporary storage in the liver and lasting storage in adipose tissue respectively. Myocytes help to lower blood glucose in response to insulin by increasing their rates of glucose uptake, glycogen synthesis, and glycolysis. The net effect of insulin is thus the replenishment of glycogen stores.

Glucagon and the fasting state

During exercise or an extended fasting or between meals, the drop in blood glucose level releases this inhibition causing the pancreatic α-cells to secrete glucagon. Glucagon stimulates glycogen breakdown in the liver in response to low blood glucose level by binding to the seven-transmembrane (7TM) receptors in the plasma membranes of hepatocytes. This binding event promotes a conformational change in the receptor’s intracellular domain that affects its interaction with the second protein in the signal-transduction pathway, a stimulatory G protein or GS, on the cytosolic side of the plasma membrane. The induced conformational change activates a GTP-binding protein Gs. The GTP-bound subunit of Gs activates the transmembrane protein adenylate cyclase (AC) which catalyzes the formation of the second messenger cyclic AMP (cAMP) from ATP. The increased [cAMP] activates PKA which mediates all the effects of glucagon. It phosphorylates phosphorylase kinase and glycogen synthase kinase triggering glycogen breakdown and blocking glycogen synthesis.

The external peptide signal is transmitted through structural changes in signaling proteins from the 7TM receptor to AC through the Gs protein. GTP-binding stimulatory G proteins that interact with serpentine receptors are heterotrimeric: trimers of three different subunits. They have at least two stable conformations: GDP-bound and GTP-bound. Interconversion between these states only occurs in a unidirectional cycle due to the irreversibility of GTP hydrolysis. Change in the conformational states of a G protein and subsequent activation is induced by interaction with a specific protein called GTPase activating protein (GAP). GAP stimulates the hydrolysis of the bound GTP by the Gs. On the other hand, in preparation for activation, the exchange of bound GDP for GTP is catalyzed by a specific guanine nucleotide exchange factor (GEF). G proteins function as molecular switches. They stimulate the production of cAMP by AC in the plasma membrane when GTP is bound but not when GDP is bound.

27 | P a g e

Glucagon receptors on liver cell surfaces respond to the presence of glucagon by activating AC and increasing the [cAMP].

The liver is more responsive to glucagon since it functions to maintain the level of blood glucose. The controlled release of glucose into the bloodstream from glycogen breakdown in the liver maintains blood-glucose concentration between meals. In liver, which does not employ glucose as a major energy source, the enzyme glucose-6-phosphatase (G6Pase) hydrolyzes G6P. The resulting glucose enters the bloodstream, thereby increasing the blood glucose concentration. However, the release of glucose is possible only in liver, because other tissues such as muscle and brain lack G6Pase so that they retain their G6P. Unlike glucose, G6P cannot pass through the cell membrane. When blood glucose levels return to normal, glucose enters pancreatic cells and hepatocytes via GLUT2. In the pancreatic α-cells, glucose inhibits the release of glucagon into the bloodstream. In hepatocytes, glucose binds to an inhibitory allosteric site on phosphorylase a facilitating the formation of the T state of phosphorylase which activates PP1.

Epinephrine and the stress response

When an animal is confronted with a stressful situation that requires increased activity—fighting or fleeing such as fear, excitement, exercise, muscular activity or its anticipation —neuronal signals from the brain trigger the release of epinephrine and norepinephrine from the adrenal medulla into the bloodstream. Epinephrine (adrenaline) and norepinephrine (noradrenaline), which are often called the “fight-or-flight” hormones, are catecholamine derivatives of tyrosine. Epinephrine binds to specific seventransmembrane (7TM) receptors in the plasma membranes of target cells. The binding events ultimately stimulate activation of two second messenger systems. Frist, activation of α-adrenergic receptors triggers the calcium-dependent phosphorylation cascade. Activated receptors stimulates phospholipase C (PLC) which hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP2) to release inositol- 2+ 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 stimulates the release of Ca from the endoplasmic reticulum, whereas DAG, together with Ca2+, activates protein kinase C (PKC). PKC requires both Ca2+ and DAG for full activity. Increasing cytosolic [Ca2+] activates phosphorylase kinase through its calmodulin subunit to stimulate glycogenolysis. It phosphorylates glycogen synthase to inhibit glycogen synthesis.

Second, activation of β-adrenergic receptors triggers the cAMP-dependent phosphorylation cascade. Activated receptors stimulate adenylate cyclase (AC) which increases the intracellular [cAMP]. The rise

28 | P a g e in [cAMP] activates PKA which phosphorylates phosphorylase b kinase to promote glycogen breakdown while inhibiting glycogen synthesis. In muscle, neuronal stimulation of muscle contraction increases cytosolic [Ca2+] reinforcing the cells’ response to cAMP. Epinephrine markedly enhances glycogen breakdown to provide fuel for glycolysis that sustains muscle contraction for the fight-or-flight response. The G6P produced by glycogen breakdown in muscle enters the glycolytic pathway; thereby generating ATP and helping the muscles cope with the stress. Insulin and epinephrine have antagonistic effects on glycogen metabolism in muscle. In liver, stress response is mediated by two types of receptors: α- adrenergic receptors which trigger calcium-dependent signaling cascade via PLC and β-adrenergic receptors which trigger cAMP-dependent phosphorylation cascade via AC.

The dual stimulation of receptors in response to epinephrine causes the liver to produce G6P, which is hydrolyzed by G6Pase, resulting in the release of glucose into the bloodstream, thereby further fueling the muscles. In the pancreatic α-cells, epinephrine stimulates to release glucagon, which further increases liver [cAMP]. Glucagon and epinephrine control both glycogen breakdown and glycogen synthesis through protein kinase A. Stimulation of glucagon receptors by glucagon also activates AC. Glucagon results in increase in liver [cAMP] and a decreased liver fructose-2,6-bisphosphate [F2,6P]. The three major enzymes phosphorylated in response to glucagon (liver) and epinephrine (liver and muscle) are phosphorylase kinase, glycogen phosphorylase a, and glycogen synthase.

The fructose-2,6-bisphosphate (F2,6P) control system

The liver β-D-Fructose-2,6-bisphosphate (F2,6P), which is not a glycolytic metabolite, is an extremely potent allosteric activator of phosphofructokinase (PFK) and an inhibitor of fructose bisphosphatase (FBPase). The cellular concentration of F2,6P depends on the balance between its rates of synthesis by phosphofructokinase-2 (PFK-2 or 6PF-2-K) and degradation by fructose bisphosphatase-2 (FBPase-2 or F-2,6-Pase). In animals, these two enzyme activities occur on different domains of the same protein. The FBPase-2 domain is structurally related to phosphoglyceratemutase (PGM) and shares a common catalytic mechanism involving a covalent phospho-His intermediate. The enzyme activities of PFK- 2/FBPase-2 are subject to covalent modification by PKA PP1. Phosphorylation at Ser-32 inhibits PFK-2 activity and activates FBPase-2 activity. The liver F2,6P control system is an important regulator of glucose secretion and maintenance of blood glucose level.

The F2,6P control systems in skeletal muscles and in heart muscle function quite differently from that in liver due to the presence in different tissue-specific PFK-2/FBPase-2 isozymes. In heart muscles,

29 | P a g e increased glycogen breakdown is coordinated with increased glycolysis rather than increased glucose secretion. Phosphorylation of heart muscle PFK-2/FBPase-2 isozyme occurs at entirely different sites from that of the liver isozyme and activates rather than inhibits PFK-2. Consequently, hormones that stimulate glycogen breakdown also increase heart muscle [F2,6P] and stimulate glycolysis. Epinephrine dilates the respiratory passages to facilitate the uptake of O2, increase the rate and strength of the heartbeat, and raise the blood pressure, thereby promoting the flow of O2 and fuels to the tissues. On the other hand, the skeletal muscle and testis isozymes lack phosphorylation sites altogether and are therefore not subjected to cAMP-dependent phosphorylation control.

Glycogen storage diseases

Type I: Glucose-6-phosphatase deficiency (von Gierke’s disease)

Type I glycogen storage disease or Von Gierke’s disease is caused by genetic defects in any of the proteins that transport G6P (T1), Pi (T2) or glucose (T3) across the endoplasmic reticulum membrane or in the protein that transports glucose across the liver cell plasma membrane (GLUT2). These proteins are important components of the G6P hydrolysis system. G6P does not leave the liver, because it cannot cross the plasma membrane. It must be transported into the lumen of the endoplasmic reticulum via T1 to be hydrolyzed by G6Pase which catalyzes the final step leading to the release of glucose into the bloodstream by the liver. Deficiency of any of the components of this hydrolytic system results in an increase of intracellular [G6P] that leads to serious derangement of glycogen metabolism.

The normal G6P hydrolysis system is missing in the liver and kidney of patients with this disease which leads to accumulation of abnormally large amounts of glycogen of normal structure in the liver and kidney. Patients who have von Gierke disease are characterized by inability to increase blood glucose concentration on administration of epinephrine and glucagon, an increased dependence on fat metabolism and failure to thrive. The presence of excess G6P in the liver triggers an increase in glycolysis, leading to a high level of lactate and pyruvate in the blood. The symptoms of Type I glycogen storage disease include huge abdomen caused by massive liver enlargement, kidney failure, severe hypoglycemia (low blood sugar), ketosis, hyperuricemia, hyperlipemia. An infant with this glycogen- storage disease may have convulsions because of the low blood-glucose level.

Type II: α-1,4-glucosidase deficiency (Pompe’s disease)

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Type II glycogen storage disease or Pompe’s disease is caused by genetic defects of the hydrolytic enzyme α-1,4-glucosidase confined to lysosomes. The α-1,4-glucosidasee enzyme hydrolyzes the disaccharide maltose, some linear oligosaccharides, and the outer branches of glycogen to produce free glucose. Deficiency of this hydrolytic enzyme results in a large accumulation of glycogen of normal structure in the lysosomes of all cells. Large accumulations of cytoplasmic glycogen of normal structure in skeletal and cardiac muscle are called glycogen lakes. Patients who have Pompe’s disease are characterized by glycogen-engorged lysosomes throughout the cell, including the myofibrils. As the disease progresses, lysosomes may rupture, releasing large amounts of glycogen into the cytoplasm. The symptoms of Type II glycogen storage disease include juvenile muscle defects (myopathy), muscular dystrophy in adults and cardiorespiratory failure. This is the most devastating glycogen storage disease. It causes death by cardiorespiratory failure.

Type III: Amylo-1,6-glucosidase deficiency (Cori’s disease)

Type III glycogen storage disease or Cori’s disease is caused by genetic defects of the hydrolytic enzyme α-1,6-glucosidase or glycogen debranching enzyme. The α-1,6-glucosidase enzyme transfers a block of three glucose residues and hydrolyzes the fourth remaining branching point to allow glycogen phosphorylase reaction to go to completion. Consequently, only the outermost branches of glycogen can be effectively utilized whereas the inner branches cannot be further degraded. Cori’s disease results in accumulation of markedly increased amount of abnormal glycogen in both liver and muscle. Remarkably, the outer branches of the glycogen are very short. Patients who have Pompe’s disease are characterized by low blood sugar due to decreased efficiency of glycogen breakdown. Type III disease cannot be distinguished from type I by physical examination alone. The hypoglycemic symptoms can be treated with frequent feeding and a high protein diet. The liver also synthesizes glucose from amino acids through gluconeogenesis in response to low blood sugar.

Type IV: Amylo (1,4→1,6)-transglycosylase deficiency (Andersen’s disease)

Type IV glycogen storage disease or Anderson’s disease is caused by genetic defects of the sugar transferring enzyme α (1,4→1,6)-transglycosylase or glycogen branching enzyme. Glycogen branching enzyme catalyzes the transfer of a terminal block of α(1→ 4)-linked 6 or 7 glucosyl residues. Deficiency of this enzyme results in a large accumulation of abnormal glycogen in both liver and muscle. Remarkably, the structure of glycogen is characterized by markedly decreased branching which greatly reduces the solubility of glycogen. The symptoms of Type IV glycogen storage disease include enlarged

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liver and spleen, progressive cirrhosis of the liver and myoglobin in urine. This is one of the most severe glycogen storage diseases; victims rarely survive past the age of 5 years because of liver dysfunction. Liver failure causes death. It has been suggested that the liver dysfunction may be caused by a “foreign body” immune reaction to the abnormal glycogen.

Type V: Muscle phosphorylase deficiency (McArdle’s disease)

Type V glycogen storage disease or McArdle’s disease is caused by genetic defects of the muscle glycogen phosphorylase enzyme which catalyzes the phosphorolytic breakdown of glycogen. Deficiency of this enzyme results in the inability of glycogen breakdown in muscle cells to provide sufficient fuel for glycolysis and keep up with the metabolic demand for ATP. McArdle’s disease is a rare inherited glycogen storage disease in which a patient’s capacity to perform strenuous exercise is limited because of painful muscle cramps. In McArdle disease, the muscle tissue is characterized by absence of any glycogen phosphorylase activity. In contrast, the liver glycogen phosphorylase is normal, implying the presence of different glycogen phosphorylase isozymes in muscle and liver. The defect in glycogen metabolism confined to muscle cells. Consequently, the symptoms of Type V glycogen storage disease include painful muscle cramps on strenuous exertion, exercise-induced cramps, pain and myoglobin in urine.

Hereditary glycogen storage diseases

Type Enzyme deficiency Tissue Common name Glycogen structure I G6Pase or transport proteins Liver and kidney von Gierke’s disease Normal but ↑ amount II α-1,4-Glucosidase All tissues Pompe’s disease Normal ↑ amount III Amylo-1,6-glucosidase All tissues Cori’s disease Short IV Amylo-(1,4→1,6)-transglycosylase All tissues Andersen’s disease Long Unbranched V Glycogen phosphorylase Muscle McArdle’s disease Normal VI Glycogen phosphorylase Liver Hers’ disease Normal VII Phosphofructokinase Muscle Tarui’s disease Normal VIII Phosphorylase kinase Liver X-Linked PK kinase D Normal IX Phosphorylase kinase All tissues Normal 0 Glycogen synthase Liver Normal

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