The Metabolism of Glycogen in Animals

CHAPTER 15 THE METABOLISM OF GLYCOGEN IN ANIMALS granules allows cells in the liver and muscle to make large numbers of glucose and glucose phosphate monomers available quickly, without raising the osmolarity of the cytosol by storing them in monomeric form. Monomers are released from glycogen granules by a phosphorolysis reaction that creates phosphorylated glucose molecules that can enter glycolysis to supply energy to the cell. Skeletal muscle cells especially require stores of glycogen to supply energy for bursts of activity. In the liver, the phosphate can be removed, allowing free glucose to be transported out of the cell to the blood for use in the brain and other tissues when dietary glucose is not sufficient. Glycogen synthesis requires a protein primer and an activated glucose precursor. Individual glucose molecules activated as sugar nucleotides are added to the nonreducing end of the growing linear chains in the outer tiers of the glycogen β -granules, and a branching enzyme adds branches periodically. Regulation of the balance between the formation of glycogen from excess glucose and the release of glucose from glycogen polymers when it is needed in metabolism is a critical function of cellular and organismal homeostasis. This balance, ultimately controlled by the hormones epinephrine, glucagon, and insulin, is achieved through allosteric regulation and phosphorylation of the synthetic and degradative enzymes. These enzymes, and the regulatory proteins that act on them, are integral parts of the glycogen granule. Glycogen was discovered in the mid-1800s by Claude Bernard. The French physiologist also found that a liver “ferment” (enzyme) released a reducing sugar from liver tissue. He named this reducing sugar matière glycogène — sugar-forming substance. In the first half of the twentieth century, scientists in laboratories around the world followed up this early work, purifying the “ferments” that synthesize and degrade glycogen and characterizing the regulation of these enzymes by insulin and epinephrine. These studies characterized the enzymes and also uncovered multiple regulatory mechanisms that proved to be universal: second messengers responsive to extracellular signals, protein kinase cascades, and protein phosphorylation, for example. In this chapter, we begin by exploring the structure and function of glycogen particles, describe the pathways of glycogen breakdown and synthesis, and finally, dig into the complex web of regulatory controls that exquisitely deliver the necessary amount of energy from glucose that each organ system requires to function in the moment. 15.1 The Structure and Function of Glycogen Structure–function relationships are key to understanding biomolecules and biochemical systems, and glycogen is no exception. The compact structure of glycogen granules allows cells to store glucose when it is available in excess, and to make it available on short notice when needed. Subtle tissue-specific differences in the enzymes (isozymes) that act on glycogen determine the dynamics of glycogen metabolism in each tissue. Vertebrate Animals Require a Ready Fuel Source for Brain and Muscle For all vertebrate animals, maintaining a ready supply of glucose for the brain and muscles is a top metabolic priority. The challenge for cells is to be able to store glucose in a form that rapidly sequesters it when the glucose concentration in the blood is high (say, aer a meal) but allows it to be accessed quickly for use particularly by the brain and skeletal muscle. Recall from our discussion in Chapter 7 (p. 242) that a typical hepatocyte in the fed state stores an amount of glucose, polymerized as glycogen, that in monomeric form would be equivalent to about 0.4 M . At this concentration, the osmolarity of the cell would be so high relative to the surrounding fluid that water would enter the cell and likely rupture it. When the diet temporarily provides more carbohydrate than is needed immediately as fuel, excess glucose is polymerized into glycogen. Small amounts of glycogen are present in all animal cells, but it is stored primarily in liver and muscle, where it is a significant portion of the wet weight of the organ (5% to 10% of liver and 1% to 2% of muscle). A 70 kg human stores about 100 g of glycogen in the liver and up to 400 g in skeletal muscle. The total amount of energy stored in the body as glycogen is far less (about 1%) than the amount stored as fat (triacylglycerol), but fats cannot be converted to glucose in vertebrates and cannot be catabolized anaerobically through glycolysis, as is oen required in skeletal muscle. When a sudden burst of physical activity demands a quick source of energy in muscle, the rapid breakdown of glycogen stored there provides glucose for glycolysis within seconds. Between meals or during a fast, the release of glucose from glycogen stored in the liver provides a steady supply of glucose in the blood. This is especially important for the brain, a major consumer of metabolic energy, which, unlike muscle, cannot use fatty acids as fuel; long-chain fatty acids do not cross the blood- brain barrier. The brain therefore depends on a constant supply of glucose, from the diet or from the liver. Glycogen Granules Have Many Tiers of Branched Chains of -Glucose Glycogen is stored as cytosolic granules, called β -granules, that vary in size, structure, and subcellular location depending on the tissue or cell type. (In this chapter we focus on liver and muscle.) The size of β -granules also varies with the state of activity and feeding of the animal. In muscle, β -granules are 20–30 nm in diameter and have an Mr of 106–107. They consist of up to 55,000 glucose residues with about 2,000 nonreducing ends available for degradative enzymes to work on. In liver, 20 to 40 β -granules cluster together to form protein-rich α -granules as large as 300 nm in diameter and of Mr greater than 108. They are visible with the electron microscope in tissue samples from well-fed animals (Fig. 15-1), but they are essentially absent aer a 24-hour fast. The β -granules of muscle release glucose more quickly than the α - granules of liver, consistent with the different needs of these tissues for glucose. FIGURE 15-1 Glycogen granules in a hepatocyte. Glycogen β -granules appear as electron-dense particles. In liver they form larger clusters called α -granules and are o en associated with tubules of the smooth endoplasmic reticulum. Four mitochondria are also evident in this micrograph. All glycogen granules have at their core a dimer of the protein glycogenin, which serves as a primer for the synthesis of polymers of D-glucose. In the tiered β -granule model, the central glycogenin dimer is surrounded by tier upon tier of chains of about 13 glucose residues in (α1→ 4) linkage, with (α1→ 6)- linked branches. Inner B-chains contain two branch points, and outer A-chains are unbranched (Fig. 15-2). Granules typically have 6 or 7 tiers, with the outermost tier of unbranched A-chains making up the majority of the granule. Associated with each β - granule are patches of electron-dense, protein-rich material, called γ -particles. Among the associated proteins are the enzymes that synthesize and break down glycogen. FIGURE 15-2 Structure of a glycogen β -granule. Starting at a central glycogenin homodimer, glycogen chains (12 to 14 residues) extend in tiers. Inner chains (B-chains) have two (α1→ 6) branches each. A-chains in the outer tier are unbranched. There are in theory a maximum of 12 tiers in a mature glycogen β -granule (only 5 are shown here), consisting of about 55,000 glucose residues in a molecule of about 21 nm diameter and Mr~1 × 107. The general mechanisms for storing and mobilizing glycogen are the same in muscle and liver, but the enzymes involved differ in subtle yet important ways that reflect the different roles of glycogen in the two tissues. In the next two sections we will look at the enzymatic basis for glycogen synthesis and breakdown, and at the regulation of these processes. SUMMARY 15.1 The Structure and Function of Glycogen All cells need ready access to glucose, either from the diet or from supplies stored in cells. Glycogen is a polymeric storage form of glucose in animals that is found primarily in muscle and liver. Glycogen breakdown in muscle delivers glucose needed for muscle contraction. Glycogen stored in the liver provides a reservoir that maintains homeostasis of blood glucose throughout the body. Glycogen β -granules have tiers of glucose residues in (α1→ 4) linkage, with (α1→ 6)-linked branches, providing many free nonreducing ends for synthetic and degradative enzymes to access. In liver, β -granules cluster into larger α -granules, which release glucose more slowly. 15.2 Breakdown and Synthesis of Glycogen As we saw in Chapter 14 (Fig. 14-9), glycogen obtained in the diet is broken down by α -amylases, hydrolytic enzymes that act in the mouth and gut to convert glycogen to free glucose. (Dietary starch is hydrolyzed in a similar way.) But glycogen stored in cells (endogenous glycogen) is degraded by a different pathway. We begin with the breakdown of cellular glycogen to glucose 1- phosphate (glycogenolysis), then turn to synthesis of glycogen (glycogenesis). Glycogen Breakdown Is Catalyzed by Glycogen Phosphorylase In skeletal muscle and liver, the glucose units of the outer branches of glycogen enter the glycolytic pathway through the action of three enzymes: glycogen phosphorylase, glycogen debranching enzyme, and phosphoglucomutase. Glycogen phosphorylase catalyzes the reaction in which an (α1→ 4) glycosidic linkage between two glucose residues at a nonreducing end of glycogen undergoes attack by inorganic phosphate (Pi), removing the terminal glucose residue as α -D-glucose 1- phosphate (Fig. 15-3). This phosphorolysis reaction is different from the hydrolysis of glycosidic bonds by amylase during intestinal degradation of dietary glycogen and starch. In phosphorolysis, some of the energy of the glycosidic bond is conserved in the formation of the phosphate ester glucose 1- phosphate. FIGURE 15-3 Removal of a glucose residue from the nonreducing end of a glycogen chain by glycogen phosphorylase. This process is repetitive; the enzyme removes successive glucose residues, creating a new nonreducing end, until it reaches the fourth glucose unit from a branch point (see Fig. 15- 4). Pyridoxal phosphate is an essential cofactor in the glycogen phosphorylase reaction. It is covalently attached near the enzyme active site, where its phosphate group acts as a general acid catalyst, promoting attack by Pi on the glycosidic bond. (This is an unusual role for pyridoxal phosphate; its more typical role is as a cofactor in amino acid metabolism; see Fig. 18-6.) Glycogen phosphorylase acts repetitively on the nonreducing ends of glycogen branches until it reaches a point four glucose residues away from an (α1→ 6) branch point, where its action stops. Further degradation by glycogen phosphorylase can occur only aer the debranching enzyme, formally known as oligo (α1→ 6) to (α1→ 4) glucantransferase, catalyzes two successive reactions that transfer branches (Fig. 15-4) to form straight chains. Once these branches are transferred and the glucosyl residue at C-6 is hydrolyzed, glycogen phosphorylase activity can continue.

FIGURE 15-4 Glycogen breakdown near an (α1→ 6) branch point. Following sequential removal of terminal glucose residues by glycogen phosphorylase (see Fig. 15-3), glucose residues near a branch are removed in a two-step process that requires a bifunctional debranching enzyme. First, the transferase activity of the enzyme shi s a block of three glucose residues from the branch to a nearby nonreducing end, to which the segment is reattached in (α1→ 4) linkage. The single glucose residue remaining at the branch point, in (α1→ 6) linkage, is then released as free glucose by the (α1→ 6) glucosidase activity of the debranching enzyme. The glucose residues are shown in shorthand form. Glucose 1-Phosphate Can Enter Glycolysis or, in Liver, Replenish Blood Glucose Glucose 1-phosphate, the end product of the glycogen phosphorylase reaction, is converted to glucose 6-phosphate by phosphoglucomutase, which catalyzes the reversible reaction: Glucose1-phosphate⇌ glucose6-phosphate Initially phosphorylated at a Ser residue, the enzyme donates its phosphoryl group to C-6 of the substrate, then accepts a phosphoryl group from C-1 (Fig. 15-5). FIGURE 15-5 Reaction catalyzed by phosphoglucomutase. The reaction begins with the enzyme phosphorylated on a Ser residue. In step , the enzyme donates its phosphoryl group (blue) to glucose 1-phosphate, producing glucose 1,6-bisphosphate. In step , the phosphoryl group at C-1 of glucose 1,6-bisphosphate (red) is transferred back to the enzyme, re-forming the phosphoenzyme and producing glucose 6-phosphate. The glucose 6-phosphate formed from glycogen in skeletal muscle can enter glycolysis and serve as an energy source to support muscle contraction. In liver, glycogen breakdown serves a different purpose: to release glucose into the blood when the blood glucose level drops, as it does between meals. This requires the enzyme glucose 6-phosphatase, present in liver and kidney but not in other tissues. The enzyme is an integral protein of the endoplasmic reticulum, with its active site on the lumenal side of the ER. Glucose 6-phosphate formed in the cytosol is transported into the ER lumen by a specific transporter (T1) (Fig. 15-6) and hydrolyzed at the lumenal surface by glucose 6-phosphatase. The resulting Pi and glucose are carried back into the cytosol by two different transporters (T2 and T3), and the glucose enters the blood via the plasma membrane transporter, GLUT2. Notice that by having the active site of glucose 6-phosphatase in the ER lumen, the cell separates this reaction from the process of glycolysis, which takes place in the cytosol and would be aborted by the action of glucose 6-phosphatase. Genetic defects in either glucose 6-phosphatase or T1 lead to serious derangement of glycogen metabolism, resulting in type Ia glycogen storage disease (Box 15-1). FIGURE 15-6 Hydrolysis of glucose 6-phosphate by glucose 6-phosphatase of the liver ER. The catalytic site of glucose 6-phosphatase faces the lumen of the ER. A glucose 6-phosphate (G6P) transporter (T1) carries the substrate from the cytosol to the lumen, where glucose 6-phosphatase releases Pi. The products, glucose and Pi, pass to the cytosol on specific transporters (T2 and T3). Glucose leaves the cell via the GLUT2 transporter in the plasma membrane. BOX 15-1 MEDICINE Carl and Gerty Cori: Pioneers in Glycogen Metabolism and Disease Much of what is written in present-day biochemistry textbooks about the metabolism of glycogen was discovered between about 1925 and 1950 by the remarkable husband and wife team of Carl F. Cori and Gerty T. Cori. Both trained in medicine in Europe at the end of World War I (she completed premedical studies and medical school in one year!). They le Europe together in 1922 to establish research laboratories in the United States, first for nine years in Buffalo, New York, at what is now the Roswell Park Comprehensive Cancer Center, then from 1931 until the end of their lives at Washington University in St. Louis. In their early physiological studies of the origin and fate of glycogen in animal muscle, the Coris demonstrated the conversion of glycogen to lactate in tissues; movement of lactate in the blood to the liver; and, in the liver, reconversion of lactate to glycogen — a pathway that came to be known as the Cori cycle (see Fig. 23-17). Pursuing these observations at the biochemical level, they showed that glycogen was mobilized in a phosphorolysis reaction catalyzed by the enzyme they discovered, glycogen phosphorylase. They identified the product of this reaction (the “Cori ester”) as glucose 1-phosphate and showed that it could be reincorporated into glycogen in the reverse reaction. Although this did not prove to be the reaction by which glycogen is synthesized in cells, it was the first in vitro demonstration of the synthesis of a macromolecule from simple monomeric subunits, and it inspired others to search for polymerizing enzymes. Arthur Kornberg, discoverer of the first DNA polymerase, said of his experience in the Coris’s lab, “Glycogen phosphorylase, not base pairing, was what led me to DNA polymerase.” FIGURE 1 The Coris in Gerty Cori’s laboratory, around 1947. Gerty Cori became interested in human genetic diseases in which too much glycogen is stored in the liver. She was able to identify the biochemical defect in several of these diseases and to show that the diseases could be diagnosed by assays of the enzymes of glycogen metabolism in small samples of tissue obtained by biopsy. Table 1 summarizes what we now know about 13 genetic diseases of this sort. TABLE 1 Glycogen Storage Diseases of Humans Type (name) Enzyme affected Primary organ/cells affected Symptoms Type 0 Glycogen synthase Liver Low blood glucose, high ketone bodies, early death Type Ia (von Gierke) Glucose 6- phosphatase Liver Enlarged liver, kidney failure Type Ib Microsomal glucose 6- phosphate translocase Liver As in type Ia; also high susceptibility to bacterial infections Type Ic Microsomal Pi transporter Liver As in type Ia Type II (Pompe) Lysosomal glucosidase Skeletal and cardiac muscle Infantile form: death by age 2; juvenile form: muscle defects (myopathy); adult form: as in muscular dystrophy Type IIIa (Cori or Forbes) Debranching enzyme Liver, skeletal and cardiac muscle Enlarged liver in infants; myopathy Type IIIb Liver debranching enzyme (muscle enzyme normal) Liver Enlarged liver in infants Type IV (Andersen) Branching enzyme Liver, skeletal muscle Enlarged liver and spleen, myoglobin in urine Type V (McArdle) Muscle phosphorylase Skeletal muscle Exercise- induced cramps and pain; myoglobin in urine Type VI (Hers) Liver phosphorylase Liver Enlarged liver Type VII (Tarui) Muscle PFK-1 Muscle, erythrocytes As in type V; also hemolytic anemia Type VIb, VIII, or IX Phosphorylase kinase Liver, leukocytes, muscle Enlarged liver Type XI (Fanconi- Bickel) Glucose transporter (GLUT2) Liver Failure to thrive, enlarged liver, rickets, kidney dysfunction Carl and Gerty Cori shared the Nobel Prize in Physiology or Medicine in 1947 with Bernardo Houssay of Argentina, who was cited for his studies of hormonal regulation of carbohydrate metabolism. The Cori laboratories in St. Louis became an international center of biochemical research in the 1940s and 1950s, and at least six scientists who trained with the Coris became Nobel laureates: Arthur Kornberg (for DNA synthesis, 1959), Severo Ochoa (for RNA synthesis, 1959), Luis Leloir (for the role of sugar nucleotides in polysaccharide synthesis, 1970), Earl Sutherland (for the discovery of cAMP in the regulation of carbohydrate metabolism, 1971), Christian de Duve (for subcellular fractionation, 1974), and Edwin Krebs (for the discovery of phosphorylase kinase, 1991). Because muscle and adipose tissue lack glucose 6-phosphatase, they cannot convert the glucose 6-phosphate formed by glycogen breakdown to glucose, and these tissues therefore do not contribute glucose to the blood. The Sugar Nucleotide UDP-Glucose Donates Glucose for Glycogen Synthesis Many of the reactions in which hexoses are transformed or polymerized involve sugar nucleotides, compounds in which the anomeric carbon of a sugar is activated by attachment to a nucleotide through a phosphate ester linkage. Sugar nucleotides are the substrates for polymerization of monosaccharides into disaccharides, glycogen, starch, cellulose, and more complex extracellular polysaccharides. They are also key intermediates in the production of the aminohexoses and deoxyhexoses found in some of these polysaccharides, and in the synthesis of vitamin C (L-ascorbic acid). The role of sugar nucleotides in the biosynthesis of glycogen and many other carbohydrate derivatives was discovered in 1953 by the Argentine biochemist Luis Leloir. The suitability of sugar nucleotides for biosynthetic reactions stems from several properties: 1. Their formation is metabolically irreversible, contributing to the irreversibility of the synthetic pathways in which they are intermediates. The condensation of a nucleoside triphosphate with a hexose 1-phosphate to form a sugar nucleotide has a small positive free-energy change, but the reaction releases PPi, which is immediately hydrolyzed by inorganic pyrophosphatase (Fig. 15-7), in a reaction that is strongly exergonic (ΔG′°=−19.2kJ /mol). This keeps the cellular concentration of PPi low, ensuring that the actual free-energy change in the cell is favorable. In effect, rapid removal of the product, driven by the large, negative free- energy change of PPi hydrolysis, pulls the synthetic reaction forward. This is a common strategy in biological polymerization reactions. 2. Although the chemical transformations of sugar nucleotides do not involve the atoms of the nucleotide itself, the nucleotide moiety has many groups that can undergo noncovalent interactions with enzymes; the additional free energy of binding can contribute significantly to catalytic activity (Chapter 6; see also p. 294). 3. Like phosphate, the nucleotidyl group (UDP or ADP, for example) is an excellent leaving group, facilitating nucleophilic attack by activating the sugar carbon to which it is attached. 4. By “tagging” some hexoses with nucleotidyl groups, cells can set them aside in a pool for a particular purpose (glycogen synthesis, for example), separate from hexose phosphates destined for another purpose (such as glycolysis). FIGURE 15-7 Formation of a sugar nucleotide. A condensation reaction occurs between a nucleoside triphosphate (NTP) and a sugar phosphate. The negatively charged oxygen on the sugar phosphate serves as a nucleophile, attacking the α phosphate of the nucleoside triphosphate and displacing pyrophosphate. The reaction is pulled in the forward direction by the hydrolysis of PPi by inorganic pyrophosphatase. Glycogen synthesis takes place in virtually all animal tissues but is especially prominent in the liver and skeletal muscles. The starting point for synthesis of glycogen is glucose 6-phosphate. This can be derived from free glucose in a reaction catalyzed by the isozymes hexokinase I and II in muscle and hexokinase IV (glucokinase) in liver: Glucose+ AT P → glucose6-phosphate+ AD P However, some ingested glucose takes a more roundabout path to glycogen. It is first taken up by erythrocytes and converted to lactate via glycolysis; the lactate is then taken up by the liver and converted to glucose 6-phosphate by gluconeogenesis. To start glycogen synthesis, the glucose 6-phosphate is converted to glucose 1-phosphate in the phosphogluco-mutase reaction: Glucose6-phosphate⇌ glucose1-phosphate The product is then converted to UDP-glucose by the action of UDP-glucose pyrophosphorylase, in a key step of glycogen biosynthesis: Glucose1-phosphate+ U T P → U D P-glucose+ PPi Notice that this enzyme is named for the reverse reaction; in the cell, the reaction proceeds in the direction of UDP-glucose formation, because the pyrophosphate concentration is kept low by its immediate hydrolysis by inorganic pyrophosphatase (Fig. 15-7). UDP-glucose is the immediate donor of glucose residues in the reaction catalyzed by glycogen synthase, which promotes the transfer of the glucose residue from UDP-glucose to a nonreducing end of a branched glycogen molecule, forming an (α1→ 4) linkage (Fig. 15-8). The overall equilibrium of the path from glucose 6-phosphate to glycogen lengthened by one glucose unit greatly favors synthesis of glycogen. FIGURE 15-8 Glycogen synthesis. A glycogen chain is elongated by glycogen synthase. The enzyme transfers the glucose residue of UDP-glucose to the nonreducing end of a glycogen branch to make a new (α1→ 4) linkage. Glycogen synthase cannot make the (α1→ 6) bonds found at the branch points of glycogen; these are formed by the glycogen- branching enzyme, also called amylo (1→ 4) to (1→ 6) transglycosylase, or glycosyl (4→ 6) transferase. The glycogen- branching enzyme catalyzes transfer of a terminal fragment of 6 or 7 glucose residues from the nonreducing end of a glycogen branch having at least 11 residues to the C-6 hydroxyl group of a glucose residue at a more interior position of the same or another glycogen chain, thus creating a new branch (Fig. 15-9). Further glucose residues may be added to the new branch by glycogen synthase. The biological effect of branching is to increase the number of nonreducing ends. This increases the number of sites accessible to glycogen phosphorylase and glycogen synthase, both of which act only at nonreducing ends. FIGURE 15-9 Branch synthesis in glycogen. The glycogen-branching enzyme forms a new branch point during glycogen synthesis. Glycogenin Primes the Initial Sugar Residues in Glycogen Glycogen synthase cannot initiate a new glycogen chain de novo. It requires a primer, usually a preformed (α1→ 4) polyglucose chain. So, how is a new glycogen molecule initiated? The intriguing protein glycogenin (Fig. 15-10) is both the primer on which new chains are assembled and the enzyme that catalyzes their assembly. The first step in the synthesis of a new glycogen molecule is the transfer of a glucose residue from UDP-glucose to the hydroxyl group of T yr194 of glycogenin. Each subunit of the glycogenin homodimer glycosylates T yr194 of the other subunit. The glycosidic bond in the product has the same configuration about the C-1 of glucose as the substrate UDP-glucose, suggesting that the transfer of glucose from UDP to T yr194 occurs in two steps. The first step is probably a nucleophilic attack by Asp162, forming a temporary intermediate with inverted configuration. A second nucleophilic attack by T yr194 then restores the starting configuration. The nascent chains are extended by the sequential addition of seven more glucose residues, each derived from UDP- glucose; the reactions are catalyzed by the chain-extending activity of glycogenin. At this point, glycogen synthase takes over, further extending the glycogen chain. Glycogenin remains buried within the β -particle, covalently attached to the two reducing ends of the glycogen molecule.

FIGURE 15-10 Glycogenin. (a) The protein is a homodimer. The substrate, UDP-glucose, is bound in a region near the amino terminus and is some distance from the Tyr194 residues — 15 Å from the Tyr in the same monomer, 12 Å from the Tyr in the dimeric partner. Each UDP-glucose is bound through its phosphates to a M n2+ ion, which is essential to catalysis. M n2+ is believed to function as an electron-pair acceptor (Lewis acid) to stabilize the leaving group, UDP. (b) Glycogenin catalyzes two distinct reactions. Initial attack by the hydroxyl group of Tyr194 on C-1 of the glucosyl moiety of UDP- glucose results in a glucosylated Tyr residue. The C-1 of another UDP-glucose molecule is now attacked by the C-4 hydroxyl group of the terminal glucose, and this sequence repeats to form a nascent glycogen molecule of eight glucose residues attached by (α1→ 4) glycosidic linkages. [(a) Data from PDB ID 1LL2, B. J. Gibbons et al., J. Mol. Biol. 319:463, 2002.] SUMMARY 15.2 Breakdown and Synthesis of Glycogen Glycogen phosphorylase catalyzes phosphorolytic cleavage at the nonreducing ends of glycogen chains, producing glucose 1- phosphate. The debranching enzyme transfers branches onto main chains and releases the residue at the (α1→ 6) branch as free glucose. Phosphoglucomutase interconverts glucose 1-phosphate and glucose 6-phosphate. Glucose 6-phosphate can enter glycolysis or, in liver, it can be converted to free glucose by glucose 6- phosphatase in the endoplasmic reticulum, then exported to replenish blood glucose. The sugar nucleotide UDP-glucose donates glucose residues to the nonreducing end of glycogen in the reaction catalyzed by glycogen synthase, producing short (α1→ 4)-linked segments. A separate branching enzyme produces the (α1→ 6) linkages at branch points. New glycogen particles begin with the autocatalytic formation of a glycosidic bond between the glucose of UDP-glucose and a Tyr residue of the protein glycogenin, followed by addition of several glucose residues to form a primer that can be acted on by glycogen synthase. 15.3 Coordinated Regulation of Glycogen Breakdown and Synthesis As we have seen, the mobilization of stored glycogen is brought about by glycogen phosphorylase, which degrades glycogen to glucose 1-phosphate (Fig. 15-3). Glycogen phosphorylase provides an especially instructive case of enzyme regulation. It was one of the first known examples of an allosterically regulated enzyme and the first enzyme shown to be controlled by reversible phosphorylation. It was also one of the first allosteric enzymes for which the detailed three-dimensional structures of the active and inactive forms were revealed by x-ray crystallographic studies. Glycogen phosphorylase also illustrates how isozymes play their tissue-specific roles. Glycogen Phosphorylase Is Regulated by Hormone-Stimulated Phosphorylation and by Allosteric Effectors Earl W. Sutherland, Jr., 1915–1974 In the late 1930s, Carl and Gerty Cori (Box 15-1) discovered that the glycogen phosphorylase of skeletal muscle exists in two interconvertible forms: glycogen phosphorylase a, which is catalytically active, and glycogen phosphorylase b, which is much less active. (Note that glycogen phosphorylase is oen referred to simply as phosphorylase — so honored because it was the first phosphorylase to be discovered; the shortened name has persisted in common usage and in the literature.) Subsequent studies by Earl Sutherland showed that phosphorylase b predominates in resting muscle, but during vigorous muscular activity, epinephrine triggers phosphorylation of phosphorylase b, converting it to its more active form, phosphorylase a (Fig. 15- 11). In the liver, glucagon triggers phosphorylation of phosphorylase b, converting it to its active form. FIGURE 15-11 Regulation of muscle glycogen phosphorylase by covalent modification. In the more active form of the enzyme, phosphorylase a, Ser14 residues, one on each subunit, are phosphorylated. Phosphorylase a is converted to the less active form, phosphorylase b, by enzymatic loss of these phosphoryl groups, catalyzed by phosphoprotein phosphatase 1 (PP1). Phosphorylase b can be reconverted (reactivated) to phosphorylase a by the action of phosphorylase b kinase. Sutherland discovered the second messenger cAMP, which increases in concentration in response to stimulation by epinephrine (in muscle; see Fig. 12-4) or glucagon (in liver). Elevated [cAMP] initiates an enzyme cascade (Fig. 15-12), in which a catalyst activates a catalyst, which activates a catalyst. As we saw in Chapter 12, such cascades allow exponential amplification of the initial signal. The rise in [cAMP] activates cAMP-dependent protein kinase, also called protein kinase A (PKA). PKA then phosphorylates and activates phosphorylase b kinase, which catalyzes the phosphorylation of glycogen phosphorylase b, activating it and thus stimulating glycogen breakdown. FIGURE 15-12 Cascade mechanism of epinephrine and glucagon action. By binding to specific surface receptors, either epinephrine acting on a myocyte (le ) or glucagon acting on a hepatocyte (right) activates a GTP- binding protein, Gsα. Active Gsα triggers a rise in [cAMP], activating PKA. This sets off a cascade of phosphorylations; PKA activates phosphorylase b kinase, which then activates glycogen phosphorylase. Such cascades effect a large amplification of the initial signal; the figures in pink boxes are certainly low estimates of the actual increase in number of molecules at each stage of the cascade. The resulting breakdown of glycogen provides glucose, which in the myocyte can supply ATP (via glycolysis) for muscle contraction and in the hepatocyte is released into the blood to counter the low blood glucose. In muscle, this provides fuel for glycolysis to sustain muscle contraction for the fight-or-flight response signaled by epinephrine. In liver, glycogen breakdown counters the low blood glucose signaled by glucagon, by releasing glucose into the blood. These different roles are reflected in subtle differences in the regulatory mechanisms in muscle and liver. The glycogen phosphorylases of liver and muscle are isozymes, encoded by different genes and differing in their regulatory properties. Figure 15-12 shows the pathway for hormonal regulation of glycogen phosphorylase activity: epinephrine or glucagon initiates the cascade that activates phosphorylase b kinase. Phosphorylase b kinase activates phosphorylase by transferring a phosphoryl group to Ser14 on each of the two identical subunits of phosphorylase b, triggering a conformation change from the T state (phosphorylase b) to the R state (phosphorylase a), shown in detail in Figure 6-40. Superimposed on the hormonal activation of phosphorylase b kinase are allosteric control mechanisms (Fig. 15-12). In muscle, Ca2+, the signal for muscle contraction, binds to and activates phosphorylase b kinase. Ca2+ binds to phosphorylase b kinase through its δ subunit, which is calmodulin (see Fig. 12-17). AMP, which accumulates in vigorously contracting muscle as a result of ATP breakdown, binds to and activates phosphorylase, speeding the release of glucose 1-phosphate from glycogen. When ATP levels are adequate, ATP blocks the allosteric site to which AMP binds (see Fig. 6-40), inactivating phosphorylase. When the muscle returns to rest, a second enzyme, phosphoprotein phosphatase 1 (PP1), removes the phosphoryl groups from phosphorylase a, converting it to the less active form, phosphorylase b (see Fig. 15-11). Like the enzyme of muscle, the glycogen phosphorylase of liver is regulated hormonally (by phosphorylation/dephosphorylation) and allosterically. The dephosphorylated form is essentially inactive. When the blood glucose level is too low, glucagon (acting through the cascade mechanism shown in Fig. 15-12) activates phosphorylase b kinase, which in turn converts phosphorylase b to its active a form, initiating the release of glucose into the blood. When blood glucose levels return to normal, glucose enters hepatocytes and binds to an inhibitory allosteric site on phosphorylase a. This binding also produces a conformational change that exposes the phosphorylated Ser residues to PP1, which catalyzes their dephosphorylation and inactivates the phosphorylase (Fig. 15-13). The allosteric site for glucose allows liver glycogen phosphorylase to act as its own glucose sensor and to respond appropriately to changes in blood glucose. FIGURE 15-13 Glycogen phosphorylase of liver as a glucose sensor. 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 phosphoprotein phosphatase 1 (PP1). This phosphatase converts phosphorylase a to phosphorylase b, sharply reducing the activity of phosphorylase and slowing glycogen breakdown in response to high blood glucose. Insulin also acts indirectly to stimulate PP1 and slow glycogen breakdown. Glycogen Synthase Also Is Subject to Multiple Levels of Regulation Like glycogen phosphorylase, glycogen synthase can exist in phosphorylated and unphosphorylated forms (Fig. 15-14). Its active form, glycogen synthase a, is unphosphorylated. Glycogen synthase kinase 3 (GSK3) adds phosphoryl groups to three Ser residues near the carboxyl terminus of glycogen synthase a converting it to glycogen synthase b, which is inactive unless its allosteric activator, glucose 6-phosphate, is present. The action of GSK3 is hierarchical; it cannot phosphorylate glycogen synthase until another protein kinase, casein kinase II (CKII), has first phosphorylated the glycogen synthase on a nearby residue, an event called priming (Fig. 15-15a). AMP-activated protein kinase (AMPK), which associates with glycogen granules through its carbohydrate-binding domain, also phosphorylates glycogen synthase, inhibiting glycogen synthesis during periods of metabolic stress, signaled by high [AMP] and low [ATP]. FIGURE 15-14 Effects of GSK3 on glycogen synthase activity. Glycogen synthase a, the active form, has three Ser residues near its carboxyl terminus. Their phosphorylation by glycogen synthase kinase 3 (GSK3) converts glycogen synthase to its inactive b form. Insulin favors the active a form of glycogen synthase by blocking the activity of GSK3 and activating phosphoprotein phosphatase 1 (PP1). In muscle, epinephrine activates PKA, which phosphorylates the glycogen-targeting protein G on a site that causes dissociation of PP1 from glycogen. Glucose 6-phosphate favors dephosphorylation of glycogen synthase by binding to it and promoting a conformation that is a good substrate for PP1. FIGURE 15-15 Priming of GSK3 phosphorylation of glycogen synthase. (a) Glycogen synthase kinase 3 first associates with its substrate (glycogen synthase) by interaction between three positively charged residues (Arg96,Arg180,Lys205) and a phosphoserine residue at position +4 in the substrate. (For orientation, the Ser or Thr residue to be phosphorylated in the substrate is assigned the index 0. Residues on the amino-terminal side of this residue are numbered −1, − 2, and so forth; residues on the carboxyl- terminal side are numbered +1, + 2, and so forth.) This association aligns the active site of the enzyme with a Ser residue at position 0, which it phosphorylates. This creates a new priming site, and the enzyme moves down the protein to phosphorylate the Ser residue at position −4, and then the Ser at −8. (b) GSK3 has a Ser residue near its amino terminus that can be phosphorylated by PKA or PKB. This produces a “pseudosubstrate” region in GSK3 that folds into the priming site and makes the active site inaccessible to another protein substrate, inhibiting GSK3 until the priming phosphoryl group of its pseudosubstrate region is removed by PP1. Other proteins that are substrates for GSK3 also have a priming site at position +4, which must be phosphorylated by another protein kinase before GSK3 can act on them. Insulin favors activation of glycogen synthase by blocking the activity of GSK3 and activating PP1. In muscle, epinephrine activates PKA, which phosphorylates the glycogen-targeting protein GM (see Fig. 15-16), a regulatory subunit of PP1 in muscle, on a site that causes dissociation of PP1 from glycogen, effectively blocking its action on glycogen synthase. In liver, conversion of glycogen synthase b to the active form is promoted by PP1, which is bound to the glycogen particle by its regulatory subunit in liver, GL. PP1 removes the phosphoryl groups from the three Ser residues phosphorylated by GSK3. Glucose 6-phosphate binds to an allosteric site on glycogen synthase, making the enzyme a better substrate for dephosphorylation by PP1 and causing its activation. By analogy with glycogen phosphorylase, which acts as a glucose sensor, glycogen synthase can be regarded as a glucose 6-phosphate sensor. In muscle, a different phosphatase may have the role played by PP1 in liver, activating glycogen synthase by dephosphorylating it. As we saw in Chapter 12, one way in which insulin triggers intracellular changes is by activating a protein kinase (PKB) that, in turn, phosphorylates and inactivates GSK3 (see Fig. 12-23). Phosphorylation of a Ser residue near the amino terminus of GSK3 converts that region of the protein to a pseudosubstrate, which folds into the site at which the priming phosphorylated Ser residue normally binds (Fig. 15-15b). This prevents GSK3 from binding the priming site of a real substrate, thereby inactivating the enzyme and tipping the balance in favor of dephosphorylation of glycogen synthase by PP1. Glycogen phosphorylase can also affect the phosphorylation of glycogen synthase: active glycogen phosphorylase directly inhibits PP1, preventing it from activating glycogen synthase (Fig. 15-14). Insulin stimulates glycogen synthesis by activating PP1 and by inactivating GSK3. This single enzyme, PP1, can remove phosphoryl groups from all three of the enzymes phosphorylated in response to glucagon (liver) and epinephrine (liver and muscle): phosphorylase kinase, glycogen phosphorylase, and glycogen synthase. The catalytic subunit of PP1 (PP1c) does not exist free in the cytosol, but is tightly bound to its target proteins through a tissue-specific regulatory subunit, one of a family of glycogen-targeting proteins that bind glycogen and each of the three enzymes (Fig. 15-16). PP1 is itself subject to covalent and allosteric regulation: it is inactivated when phosphorylated by PKA and is allosterically activated by glucose 6-phosphate. FIGURE 15-16 Glycogen-targeting protein G. G is a regulatory subunit of PP1 in muscle, and one of a family of glycogen-targeting proteins that serve as a scaffold, binding other proteins (including PP1) to glycogen particles. G can be phosphorylated at two different sites in response to insulin or epinephrine. Insulin-stimulated phosphorylation of G site 1 activates PP1, which dephosphorylates phosphorylase kinase, glycogen phosphorylase, and glycogen synthase. Epinephrine-stimulated phosphorylation of G site 2 by PKA causes dissociation of PP1 from the glycogen particle, preventing its access to glycogen phosphorylase and glycogen synthase. PKA also phosphorylates a protein (inhibitor 1) that, when phosphorylated, inhibits PP1. By these means, insulin stimulates glycogen synthesis and inhibits glycogen breakdown, whereas epinephrine (or glucagon in the liver) has the opposite effects. Allosteric and Hormonal Signals Coordinate Carbohydrate Metabolism Globally Having looked at the mechanisms that regulate individual enzymes, we can now consider the overall shis in carbohydrate metabolism that occur in the well-fed state, during fasting, and in the fight-or-flight response — signaled by insulin, glucagon, and epinephrine, respectively. We need to contrast two cases in which regulation serves different ends: (1) the role of hepatocytes in supplying glucose to the blood; and (2) the selfish use of carbohydrate fuels by extrahepatic tissues, typified by skeletal muscle (myocytes), to support their own activities. Aer ingestion of a carbohydrate-rich meal, the elevation of blood glucose triggers insulin release (Fig. 15-17, top). In a hepatocyte, insulin has two immediate effects: it inactivates GSK3; and it activates a protein phosphatase, probably PP1. These two actions fully activate glycogen synthase. PP1 also inactivates glycogen phosphorylase a and phosphorylase kinase by dephosphorylating both, effectively stopping glycogen breakdown. Glucose enters the hepatocyte through the high-capacity transporter GLUT2, always present in the plasma membrane, and the elevated intracellular glucose leads to dissociation of hexokinase IV (glucokinase) from its nuclear regulatory protein (Fig. 14-21). Hexokinase IV enters the cytosol and phosphorylates glucose, stimulating glycolysis and supplying the precursor for glycogen synthesis. Under these conditions, hepatocytes use the excess glucose in the blood to synthesize glycogen, up to the limit of about 10% of the total weight of the liver.

FIGURE 15-17 Regulation of carbohydrate metabolism in the liver. Colored arrows indicate causal relationships between the changes they connect. For example, an arrow from ↓A to ↑B means that a decrease in A causes an increase in B. Red arrows connect events that result from high blood glucose; blue arrows connect events that result from low blood glucose. Between meals, or during an extended fast, the blood glucose level drops, triggering the release of glucagon, which, acting through the cascade shown in Figure 15-12, activates PKA. PKA mediates all the effects of glucagon (Fig. 15-17, bottom). It phosphorylates phosphorylase kinase, activating it and leading to the activation of glycogen phosphorylase. It phosphorylates glycogen synthase, inactivating it and blocking glycogen synthesis. It phosphorylates PFK-2/FBPase-2, leading to a drop in the concentration of the regulator fructose 2,6-bisphosphate, which has the effect of inactivating the glycolytic enzyme PFK-1 and activating the gluconeogenic enzyme FBPase-1 (see Fig. 14- 24). And it phosphorylates and inactivates the glycolytic enzyme pyruvate kinase. Under these conditions, the liver produces glucose 6-phosphate by glycogen breakdown and by gluconeogenesis, and it stops using glucose to fuel glycolysis or make glycogen, maximizing the amount of glucose it can release to the blood. This release of glucose is possible only in liver and kidney, because other tissues lack glucose 6-phosphatase (Fig. 15- 6). The physiology of skeletal muscle differs from that of liver in three ways important to our discussion of metabolic regulation (Fig. 15-18): (1) muscle uses its stored glycogen only for its own needs; (2) as it goes from rest to vigorous contraction, muscle undergoes very large changes in its demand for ATP, which is provided by glycolysis; (3) muscle lacks the enzymatic machinery for gluconeogenesis. The regulation of carbohydrate metabolism in muscle reflects these differences from liver. First, myocytes lack receptors for glucagon, which are present on hepatocytes. Second, 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. When epinephrine is released into the blood in a fight-or-flight situation, PKA is activated by the rise in [cAMP] and phosphorylates and activates glycogen phosphorylase kinase. The resulting phosphorylation and activation of glycogen phosphorylase results in faster glycogen breakdown. Epinephrine is not released under low-stress conditions, but with each neuronal stimulation of muscle contraction, cytosolic [Ca2+] rises briefly and activates phosphorylase kinase through its calmodulin subunit. FIGURE 15-18 Difference in the regulation of carbohydrate metabolism in liver and muscle. In liver, either glucagon (indicating low blood glucose) or epinephrine (signaling the need to fight or flee) has the effect of maximizing the output of glucose into the blood. In muscle, epinephrine increases glycogen breakdown and glycolysis, which together provide fuel to produce the ATP needed for muscle contraction. Elevated insulin triggers increased glycogen synthesis in myocytes by activating PP1 and inactivating GSK3. Unlike hepatocytes, myocytes have a reserve of the glucose transporter GLUT4 sequestered in intracellular vesicles. Insulin triggers their movement to the plasma membrane (see Fig. 12-23), where they allow increased glucose uptake. In response to insulin, therefore, myocytes help to lower blood glucose by increasing their rates of glucose uptake, glycogen synthesis, and glycolysis. Carbohydrate and Lipid Metabolism Are Integrated by Hormonal and Allosteric Mechanisms As complex as the regulation of carbohydrate metabolism is, it is far from the whole story of fuel metabolism. The metabolism of fats and fatty acids is very closely tied to that of carbohydrates. Hormonal signals such as insulin and changes in diet or exercise are equally important in regulating fat metabolism and integrating it with that of carbohydrates. We return to this overall metabolic integration in mammals in Chapter 23, aer first considering the metabolic pathways for fats and amino acids (Chapters 17 and 18). The message we wish to convey here is that metabolic pathways are overlaid with complex regulatory controls that are exquisitely sensitive to changes in metabolic circumstances. These mechanisms act to adjust the flow of metabolites through various metabolic pathways, as needed by the cell and organism, without causing major changes in the concentrations of intermediates shared with other pathways. SUMMARY 15.3 Coordinated Regulation of Glycogen Breakdown and Synthesis Glycogen phosphorylase is activated in response to epinephrine (in muscle) or glucagon (in liver), which raise [cAMP] and thereby activate PKA. PKA phosphorylates and activates phosphorylase kinase, which converts glycogen phosphorylase b to its active a form. In muscle, Ca2+ and AMP act allosterically to amplify the activity of the enzymes in this cascade. In liver, glucose acts allosterically to make phosphorylase a more susceptible to dephosphorylation/inactivation by phosphoprotein phosphatase 1 (PP1). Glycogen synthase a is inactivated by phosphorylation, ultimately catalyzed by GSK3, but primed by phosphorylation by other kinases. Insulin, by causing inactivation of GSK3 and activating PP1, favors glycogen synthase a and stimulates glycogen synthesis. Glucose 6-phosphate acts allosterically to make glycogen synthase b a better substrate for PP1, which dephosphorylates it to its active a form. In liver, glucagon stimulates glycogen breakdown and gluconeogenesis while blocking glycolysis, thereby sparing glucose for export to the brain and other tissues. In muscle, epinephrine stimulates glycogen breakdown and glycolysis, providing ATP to support contraction. Further hormonal and allosteric mechanisms regulate the use of carbohydrates and lipids as metabolic fuels. Chapter Review KEY TERMS Terms in bold are defined in the glossary. glycogenolysis glycogenesis debranching enzyme phosphoglucomutase sugar nucleotides UDP-glucose pyrophosphorylase glycogen-branching enzyme glycogenin glycogen phosphorylase a glycogen phosphorylase b enzyme cascade phosphorylase b kinase phosphoprotein phosphatase 1 (PP1) glycogen synthase a glycogen synthase kinase 3 (GSK3) glycogen synthase b casein kinase II (CKII) priming glycogen-targeting proteins PROBLEMS 1. Glycogen as Energy Storage: How Long Can a Game Bird Fly? Since ancient times, people have observed that certain game birds, such as grouse, quail, and pheasants, fatigue easily. The Greek historian Xenophon wrote: “The bustards … can be caught if one is quick in starting them up, for they will fly only a short distance, like partridges, and soon tire; and their flesh is delicious.” The flight muscles of game birds rely almost entirely on the use of glucose 1-phosphate to drive ATP synthesis (Chapter 14). The glucose 1-phosphate derives from the breakdown of stored muscle glycogen, catalyzed by the enzyme glycogen phosphorylase. The rate of ATP production is limited by the rate at which glycogen can be broken down. During a “panic flight,” the game bird’s rate of glycogen breakdown is quite high, approximately 120μm ol/m in of glucose 1-phosphate produced per gram of fresh tissue. Given that the flight muscles usually contain about 0.35% glycogen by weight, calculate how long a game bird can fly. (Assume the average molecular weight of a glucose residue in glycogen is 162 g/m ol.) 2. Enzyme Activity and Physiological Function The Vm ax of the glycogen phosphorylase from skeletal muscle is much greater than the Vm ax of the same enzyme from liver tissue. a. What is the physiological function of glycogen phosphorylase in skeletal muscle? In liver tissue? b. Why does the Vm ax of the muscle enzyme need to be greater than that of the liver enzyme? 3. Glycogen Phosphorylase Equilibrium Glycogen phosphorylase catalyzes the removal of glucose from glycogen. The Δ G′° for this reaction is 3.1 kJ/mol. a. Calculate the ratio of [Pi] to [glucose 1-phosphate] when the reaction is at equilibrium. (Hint: The removal of glucose units from glycogen does not change the glycogen concentration.) b. The measured ratio [Pi]/[glucose1-phosphate] in myocytes under physiological conditions is more than 100:1. What does this indicate about the direction of metabolite flow through the glycogen phosphorylase reaction in muscle? c. Why are the equilibrium and physiological ratios different? What is the possible significance of this difference? 4. Regulation of Glycogen Phosphorylase In muscle tissue, the rate of conversion of glycogen to glucose 6-phosphate is determined by the ratio of phosphorylase a (active) to phosphorylase b (less active). Determine what happens to the rate of glycogen breakdown if a broken cell extract of muscle containing glycogen phosphorylase is treated with (a) phosphorylase kinase and ATP (b) PP1 (c) epinephrine. 5. Glycogen Breakdown in Rabbit Muscle The intracellular use of glucose and glycogen is tightly regulated at four points. To compare the regulation of glycolysis when oxygen is plentiful and when it is depleted, consider the utilization of glucose and glycogen by rabbit leg muscle in two physiological settings: a resting rabbit, with low ATP demands, and a rabbit that sights its mortal enemy, the coyote, and dashes into its burrow. For each setting, determine the relative levels (high, intermediate, or low) of AMP, ATP, citrate, and acetyl-CoA and describe how these levels affect the flow of metabolites through glycolysis by regulating specific enzymes. (Hint: In periods of stress, rabbit leg muscle produces much of its ATP by anaerobic glycolysis (lactate fermentation) and very little by oxidation of acetyl-CoA derived from fat breakdown.) 6. Glycogen Breakdown in Migrating Birds Unlike a rabbit, running all-out for a few moments to escape a predator, migratory birds require energy for extended periods of time. For example, ducks generally fly several thousand miles during their annual migration. The flight muscles of migratory birds have a high oxidative capacity and obtain the necessary ATP through the oxidation of acetyl-CoA (obtained from fats) via the citric acid cycle. Compare the regulation of muscle glycolysis during short-term intense activity, as in a fleeing rabbit, and during extended activity, as in a migrating duck. Why must the regulation in these two settings be different? 7. Enzyme Defects in Carbohydrate Metabolism Consider the four clinical case studies, A through D. For each case, determine which enzyme is defective and designate the appropriate treatment from the lists provided at the end of the problem. Justify your choices. Answer the questions contained in each case study. (You may need to refer to information in Chapter 14.) Case A: The patient develops vomiting and diarrhea shortly aer milk ingestion. The physician administers a lactose tolerance test. (The patient ingests a standard amount of lactose, and the physician measures the glucose and galactose concentrations in his blood plasma at intervals. In individuals with normal carbohydrate metabolism, the levels increase to a maximum in about 1 hour, then decline.) The patient’s blood glucose and galactose concentrations do not increase during the test. Why do blood glucose and galactose increase and then decrease during the test in healthy individuals? Why do they fail to rise in the patient? Case B: The patient develops vomiting and diarrhea aer ingestion of milk. Blood tests show a low concentration of glucose but a much higher than normal concentration of reducing sugars. The urine tests positive for galactose. Why is the concentration of reducing sugar in the blood high? Why does galactose appear in the urine? Case C: The patient complains of painful muscle cramps when performing strenuous physical exercise but has no other symptoms. A muscle biopsy indicates a muscle glycogen concentration much higher than normal. Why does glycogen accumulate? Case D: The patient is lethargic, her liver is enlarged, and a biopsy of the liver shows large amounts of excess glycogen. She also has a lower than normal blood glucose level. What is the reason for the low blood glucose in this patient? Defective Enzyme a. Muscle PFK-1 b. Phosphomannose isomerase c. Galactose 1-phosphate uridylyltransferase d. Liver glycogen phosphorylase e. Triose kinase f. Lactase in intestinal mucosa g. Maltase in intestinal mucosa h. Muscle debranching enzyme Treatment 1. Jogging 5 km each day 2. Fat-free diet 3. Low-lactose diet 4. Avoiding strenuous exercise 5. Large doses of niacin (the precursor of NAD) 6. Frequent feedings (smaller portions) of a normal diet 8. Effects of Insufficient Insulin in a Person with Diabetes A man with insulin-dependent diabetes is brought to the hospital emergency department in a near-comatose state. While vacationing in an isolated place, he lost his insulin medication and has not taken any insulin for two days. a. For each tissue listed at the end of the problem, is each pathway faster, slower, or unchanged in this patient, compared with the normal level when he is getting appropriate amounts of insulin? b. For each pathway, describe at least one control mechanism responsible for the change you predict. Tissue and Pathways 1. Adipose: fatty acid synthesis 2. Muscle: glycolysis; fatty acid synthesis; glycogen synthesis 3. Liver: glycolysis; gluconeogenesis; glycogen synthesis; fatty acid synthesis; pentose phosphate pathway 9. Blood Metabolites in Insulin Insufficiency For the patient described in Problem 8, predict the levels of each listed metabolite in his blood before treatment in the emergency room, relative to levels maintained during adequate insulin treatment: (a) glucose (b) ketone bodies (c) free fatty acids. 10. Metabolic Effects of Mutant Enzymes Predict and explain the effect on glycogen metabolism of each of the listed defects caused by mutation: (a) Loss of the cAMP- binding site on the regulatory subunit of protein kinase A (PKA) (b) Loss of the protein phosphatase inhibitor (inhibitor 1 in Fig. 15-16) (c) Overexpression of phosphorylase b kinase in liver (d) Defective glucagon receptors in liver. 11. Hormonal Control of Metabolic Fuel Between your evening meal and breakfast, your blood glucose drops and your liver becomes a net producer rather than consumer of glucose. Describe the hormonal basis for this switch, and explain how the hormonal change triggers glucose production by the liver. 12. Altered Metabolism in Genetically Manipulated Mice Researchers can manipulate the genes of a mouse so that a single gene in a single tissue either produces an inactive protein (a “knockout” mouse) or produces a protein that is always (constitutively) active. What effects on metabolism would you predict for mice with the listed genetic changes? (a) Knockout of glycogen debranching enzyme in the liver (b) Knockout of hexokinase IV in liver (c) Knockout of FBPase-2 in liver (d) Constitutively active FBPase-2 in liver (e) Constitutively active AMPK in muscle (f) Constitutively active ChREBP in liver (see Fig. 14-28) DATA ANALYSIS PROBLEM 13. Optimal Glycogen Structure Muscle cells need rapid access to large amounts of glucose during heavy exercise. This glucose is stored in liver and skeletal muscle in polymeric form as particles of glycogen. The typical glycogen β -particle contains about 55,000 glucose residues (see Fig. 15-2). Meléndez-Hevia, Waddell, and Shelton (1993) explored some theoretical aspects of the structure of glycogen, as described in this problem. a. The cellular concentration of glycogen in liver is about 0.01μM . What cellular concentration of free glucose would be required to store an equivalent amount of glucose? Why would this concentration of free glucose present a problem for the cell? Glucose is released from glycogen by glycogen phosphorylase, an enzyme that can remove glucose molecules, one at a time, from one end of a glycogen chain (see Fig. 15-3). Glycogen chains are branched (see Fig. 15-2), and the degree of branching — the number of branches per chain — has a powerful influence on the rate at which glycogen phosphorylase can release glucose. b. Why would a degree of branching that was too low (i.e., below an optimum level) reduce the rate of glucose release? (Hint: Consider the extreme case of no branches in a chain of 55,000 glucose residues.) c. Why would a degree of branching that was too high also reduce the rate of glucose release? (Hint: Think of the physical constraints.) Meléndez-Hevia and colleagues did a series of calculations and found that two branches per chain (see Fig. 15-2) was optimal for the constraints described above. This is what is found in glycogen stored in muscle and liver. To determine the optimum number of glucose residues per chain, Meléndez-Hevia and coauthors considered two key parameters that define the structure of a glycogen particle: t= thenum beroftiers ofglucosechainsinaparticle (the mole-cule in Fig. 15-2 has five tiers); gc = thenum berofglucose residuesineachchain. The y set out to find the values of t and gc that would maximize three quantities: (1) the amount of glucose stored in the particle (GT ) per unit volume; (2) the number of unbranched glucose chains (CA) per unit volume (i.e., number of A chains in the outermost tier, readily accessible to glycogen phosphorylase); and (3) the amount of glucose available to phosphorylase in these unbranched chains (GPT ). d. Show that CA = 2t−1. This is the number of chains available to glycogen phosphorylase before the action of the debranching enzyme. e. Show that CT, the total number of chains in the particle, is given by CT = 2t− 1. For purposes of this calculation, consider the primers to be a single chain. Thus GT = gc(CT )= gc(2t− 1), the total number of glucose residues in the particle. f. Glycogen phosphorylase cannot remove glucose from glycogen chains that are shorter than five glucose residues. Show that GPT = (gc− 4)(2t−1). This is the amount of glucose readily available to glycogen phosphorylase. g. Based on the size of a glucose residue and the location of branches, the thickness of one tier of glycogen is 0.12gcnm + 0.35nm. Show that the volume of a particle, Vs, is given by the equation Vs= 4/3πt3(0.12gc+ 0.35)3nm 3. Meléndez-Hevia and coauthors then determined the optimum values of t and gc — those that gave the maximum value of a quality function, f, that maximizes GT, CA, and GPT, while minimizing Vs:  f= . They found that the optimum value of gc is independent of t. h. Choose a value of t between 5 and 15 and find the optimum value of gc. How does this compare with the gc found in liver glycogen (see Fig. 15-2)? (Hint: You may find it useful to use a spreadsheet program.) Reference Meléndez-Hevia, E., T.G. Waddell, and E.D. Shelton. 1993. Optimization of molecular design in the evolution of metabolism: the glycogen molecule. Biochem. J. 295:477–483. GT CAGPT Vs