Glycogenolysis and Glycogenesis
Page 312 CLINICAL CORRELATION 7.10 Hypoglycemia and Alcohol Intoxication Consumption of alcohol, especially by an undernourished person, can cause hypoglycemia. The same effect can result from drinking alcohol after strenuous exercise. In both cases the hypoglycemia results from the inhibitory effects of alcohol on hepatic gluconeogenesis and thus occurs under circumstances of hepatic glycogen depletion. The problem is caused by the NADH produced during the metabolism of alcohol. The liver simply cannot handle the reducing equivalents provided by ethanol oxidation fast enough to prevent metabolic derangements. The extra reducing equivalents block the conversion of lactate to glucose and promote the conversion of alanine into lactate, resulting in considerable lactate accumulation in the blood. Since lactate has no place to go, lactic acidosis (see Clin. Corr. 7.5) can develop, although it is usually mild. Low doses of alcohol cause impaired motor and intellectual performance; high doses have a depressant effect that can lead to stupor and anesthesia. Low blood sugar can contribute to these undesirable effects of alcohol. What is more, a patient may be thought to be inebriated when in fact the patient is suffering from hypoglycemia that may lead to irreversible damage to the central nervous system. Children are highly dependent on gluconeogenesis while fasting, and accidental ingestion of alcohol by a child can produce severe hypoglycemia (see Clin. Corr. 7.9). Krebs, H. A., Freedland, R. A., Hems, R., and Stubbs, M. Inhibition of hepatic gluconeogenesis by ethanol. Biochem. J. 112:117, 1969; and Service, F. J. Hypoglycemia. Med. Clin. North Am. 79:1, 1995. then phosphorylates a protein called the cAMPresponse element binding protein (CREB), a transacting factor that in its phosphorylated form can bind to a cAMPresponse element (CRE), a cis acting element within the regulatory region of genes that respond to cAMP. This promotes transcription of genes encoding key gluconeogenic enzymes such as PEP carboxykinase (Figure 7.46). By a similar mechanism, but one that causes repression of gene transcription, glucagon acts to decrease the amounts of glucokinase, 6phosphofructo1kinase, and pyruvate kinase. Insulin opposes the action of glucagon (Figure 7.46), acting through a signal cascade that results in activation of an insulinresponse element binding protein (IREB), which inhibits transcription of genes encoding key gluconeogenic enzymes by binding to an insulinresponse element (IRE) in the regulatory region of such genes. When glucose synthesis is not needed, synthesis of key gluconeogenic enzymes is turned off and synthesis of key glycolytic enzymes is turned on as a consequence of a decrease in the blood glucagon/insulin ratio. Ethanol Ingestion Inhibits Gluconeogenesis Ethanol inhibits gluconeogenesis by liver (see Clin. Corr. 7.10). It is oxidized primarily in liver with production of a large load of reducing equivalents that must be transported into the mitochondria by the malateaspartate shuttle. This excess NADH in the cytosol creates problems for liver gluconeogenesis because it forces the equilibrium of the lactate dehydrogenase and malate dehydrogenasecatalyzed reactions in the directions of lactate and malate formation, respectively: or Forcing these reactions in the directions shown above inhibits glucose synthesis by limiting the amounts of pyruvate and oxaloacetate available for the reactions catalyzed by pyruvate carboxylase and PEP carboxykinase, respectively. 7.6— Glycogenolysis and Glycogenesis Glycogen, a Storage Form of Glucose, Is Required as a Ready Source of Energy Glycogenolysis refers to breakdown of glycogen to glucose or glucose 6phosphate; and glycogenesis refers to synthesis of glycogen. These processes are of some importance in almost every tissue but especially in muscle and liver. The liver has tremendous capacity for storing glycogen. In the wellfed human, liver glycogen content can account for as much as 10% of wet weight of this organ. Muscle stores less when expressed on the same basis–a maximum of only 1–2% of its wet weight. However, since the average person has more muscle than liver, there is about twice as much total muscle glycogen as liver glycogen. Page 313 Muscle and liver glycogen stores serve completely different roles. Glycogen serves as a fuel reserve for the synthesis of ATP within muscle, whereas liver glycogen functions as a glucose reserve for the maintenance of blood glucose concentrations. Liver glycogen levels vary greatly in response to the intake of food, accumulating to high levels shortly after a meal and then decreasing slowly as it is mobilized to help maintain a nearly constant blood glucose level (see Figure 7.47). Liver glycogen is called into play between meals and to a greater extent during the nocturnal fast. In both humans and the rat, the store of liver glycogen lasts somewhere between 12 and 24 h during fasting, depending greatly, of course, on whether the individual under consideration is caged or running wild. Muscle glycogen is a source of ATP for increased muscular activity. Most of the glucose of glycogen is consumed within muscle cells without formation of free glucose as an intermediate. However, because of a special feature of glycogen catabolism to be discussed below, about 8% of muscle glycogen is converted into free glucose within the tissue. Some of this glucose may be released into the bloodstream, but most gets metabolized by glycolysis in muscle. Since muscle lacks glucose 6 phosphatase, and most free glucose formed during glycogen breakdown is further catabolized, muscle glycogen is not of quantitative importance in maintenance of blood glucose levels in the fasting state. Liver glycogen converted to glucose by glycogenolysis and glucose 6phosphatase is of much greater importance as a source of blood glucose in the fasting state. Conversion of glucose to glycogen in muscle plays an important role in lowering blood glucose levels elevated by a high carbohydrate meal. Glycogenesis in liver contributes to the lowering of blood glucose but is of less importance than glycogen synthesis in muscle. Exercise of a muscle triggers mobilization of muscle glycogen for formation of ATP. The yield of ATP and the fate of the carbon of glycogen depend on whether a ''white" or "red" muscle is under consideration. Red muscle fibers are supplied with a rich blood flow, contain large amounts of myoglobin, and are packed with mitochondria. Glycogen mobilized within these cells is converted into pyruvate, which, because of the availability of O2 and mitochondria, can be converted into CO2 and H2O. In contrast, white muscle fibers have a poorer blood supply and fewer mitochondria. Glycogenolysis within this tissue supplies substrate for glycolysis, with the end product being primarily lactate. White muscle fibers have enormous capacity for glycogenolysis and glycolysis, much more than red muscle fibers. Since their glycogen stores are limited, however, muscles of this type can only function at full capacity for relatively short periods of time. Breast muscle and the heart of chicken are good examples of white and red muscles, respectively. The heart has to beat Figure 7.47 Variation of liver glycogen levels between meals and during the nocturnal fast. Page 314 continuously and has many mitochondria and a rich supply of blood via the coronary arteries. The heart stores glycogen to be used when a greater work load is imposed. Breast muscle of chicken is not continuously carrying out work. Its important function is to enable the chicken to fly rapidly for short distances, as in fleeing from predators (or amorous roosters). Because glycogen can be mobilized so rapidly, breast muscle is designed for maximal activity for a relatively short period of time. Although it was easy to point out readily recognizable white and red muscles in the chicken, most skeletal muscles of the human body are composed of a mixture of red and white fibers in order to provide for both rapid and sustained muscle activity. The distribution of white and red muscle fibers in cross sections of a human skeletal muscle can be shown by using special staining procedures (see Figure 7.48). Glycogen granules are abundant in liver of the wellfed animal but are virtually absent from liver of the 24hfasted animal (Figure 7.49). Heavy exercise causes the same loss of glycogen granules in muscle fibers. These granules of glycogen correspond to clusters of glycogen molecules, the molecular weights of which can approach 2 × 107 Da. Glycogen is composed entirely of glucosyl residues, the majority of which are linked together by a 1,4glycosidic linkages (Figure 7.50). Branches also occur in the glycogen molecule, however, because of frequent a 1,6glycosidic linkages (Figure 7.50). A limb of the glycogen "tree" (see Figure 7.51) is characterized by branches at every fourth glucosyl residue within the more central core of the molecule. These branches occur much less frequently in outer regions of the molecule. An interesting question, which we shall attempt to answer below, is why this polymer is constructed with so many intricate branches and loose ends. Glycogen certainly stands in contrast to proteins and nucleic acids in this regard but, of course, it is a storage form of fuel and never has to catalyze a reaction or convey information within a cell. Glycogen Phosphorylase Catalyzes the First Step in Glycogen Degradation Glycogen phosphorylase catalyzes phosphorolysis of glycogen, a reaction in which Pi is used in the cleavage of an a 1,4glycosidic linkage to yield glucose 1 phosphate (Figure 7.52). This always occurs at a terminal, nonreducing end of a glycogen molecule: The reaction catalyzed by glycogen phosphorylase should be distinguished from that catalyzed by a amylase, which degrades glycogen and starch in the gut (see Chapter 26). a Amylase acts by simple hydrolysis, using water rather than inorganic phosphate to cleave a 1,4glycosidic bonds. Glycogen may contain up to 100,000 glucose residues; its structure is usually abbreviated (glucose)n. The reaction catalyzed by glycogen phosphorylase is written as The next step of glycogen degradation is catalyzed by phosphoglucomutase: This is a nearequilibrium reaction under intracellular conditions, allowing it to function in both glycogen degradation and synthesis. Like phosphoglycerate mutase (see p. 277), a bisphosphate compound is an obligatory interme Page 315 Figure 7.48 Cross section of human skeletal muscle showing red and white muscle fibers. Sections were stained for NADH diaphorase activity in (a) for ATPase activity in (b). The red fibers are dark and the white fibers are light in (a); vice versa in (b). Pictures generously provided by Dr. Michael H. Brooke of the Jerry Lewis Neuromuscular Research Center, St. Louis, Missouri. Figure 7.49 Electron micrographs showing glycogen granules (darkly stained material) in the liver of a wellfed rat (a) and the relative absence of such granules in the liver of a rat starved for 24 h (b). Micrographs generously provided by Dr. Robert R. Cardell of the Department of Anatomy at the University of Cincinnati. Page 316 Figure 7.50 Two types of linkage between glucose molecules are present in glycogen. diate: A catalytic amount of glucose 1,6bisphosphate must be present for the reaction to occur. It is produced in small quantities for this specific purpose by an enzyme called phosphoglucokinase: The next enzyme involved in glycogenolysis depends on the tissue under consideration (Figure 7.52). In liver, glucose 6phosphate produced by glycogenolysis is hydrolyzed by glucose 6phosphatase to give free glucose: Lack of this enzyme or of the translocase that transports G6P into the endoplasmic reticulum (Figure 7.37) results in type 1 glycogen storage disease (see Clin. Corr. 7.11). The overall balanced equation for removal of one glucosyl residue from glycogen in liver by glycogenolysis is then In other words, glycogenolysis in liver involves phosphorolysis but, because the phosphate ester is cleaved by a phosphatase, the overall reaction adds up to hydrolysis of glycogen. No ATP is used or formed in glycogenolysis. Figure 7.51 The branched structure of glycogen. In peripheral tissues the G6P generated by glycogenolysis is used by glycolysis, leading primarily to the generation of lactate in white muscle fibers and primarily to complete oxidation to CO2 in red muscle fibers. Since no ATP had to be invested to produce G6P obtained from glycogen, the overall equation Figure 7.52 Glycogenolysis and the fate of glycogen degraded in liver versus its fate in peripheral tissues. Page 317 CLINICAL CORRELATION 7.11 Glycogen Storage Diseases There are a number of wellcharacterized glycogen storage diseases, all due to inherited defects of one or more of the enzymes involved in the synthesis and degradation of glycogen. The liver is usually the tissue most affected, but heart and muscle glycogen metabolism can also be defective. Chen, Y. T., and Burchell, A. Glycogen storage diseases. In C. R. Scriver, A. L. Beaudet, W. S. Sly, and D. Valle (Eds.), The Metabolic and Molecular Bases of Inherited Disease, 7th ed. New York: McGrawHill, 1995, pp. 935–965. Von Gierke's Disease The most common glycogen storage disease, referred to as type I or von Gierke's disease, is caused by a deficiency of liver, intestinal mucosa, and kidney glucose 6 phosphatase. Thus diagnosis by small bowel biopsy is possible. Patients with this disease can be further subclassified into those lacking the glucose 6phosphatase enzyme per se (type Ia) and those lacking the glucose 6phosphatase translocase (type Ib) (see Figure 7.37). A genetic abnormality in glucose 6phosphate hydrolysis occurs in only about 1 person in 200,000 and is transmitted as an autosomal recessive trait. Clinical manifestations include fasting hypoglycemia, lactic acidemia hyperlipidemia, and hyperuricemia with gouty arthritis. The fasting hypoglycemia is readily explained as a consequence of the glucose 6phosphatase deficiency, the enzyme required to obtain glucose from liver glycogen and gluconeogenesis. The liver of these patients does release some glucose by the action of the glycogen debranching enzyme. The lactic acidemia occurs because the liver cannot use lactate effectively for glucose synthesis. In addition, the liver inappropriately produces lactic acid in response to glucagon. This hormone should trigger glucose release without lactate production; however, the opposite occurs because of the lack of glucose 6phosphatase. Hyperuricemia results from increased purine degradation in the liver; hyperlipidemia results because of increased availability of lactic acid for lipogenesis and lipid mobilization from the adipose tissue caused by high glucagon levels in response to hypoglycemia. The manifestations of von Gierke's disease can greatly be diminished by providing carbohydrate throughout the day to prevent hypoglycemia. During sleep this can be done by infusion of carbohydrate into the gut by a nasogastric tube. Cori, G. T., and Cori, C. F. Glucose6phosphatase of the liver in glycogen storage disease. J. Biol. Chem. 199:661, 1952. Pompe's Disease Type II glycogen storage disease or Pompe's disease is caused by the absence of a 1,4 glucosidase (or acid maltase), an enzyme normally found in lysosomes. The absence of this enzyme leads to the accumulation of glycogen in virtually every tissue. This is somewhat surprising, but lysosomes take up glycogen granules and become defective with respect to other functions if they lack the capacity to destroy the granules. Because other synthetic and degradative pathways of glycogen metabolism are intact, metabolic derangements such as those in von Gierke's disease are not seen. The reason for extralysosomal glycogen accumulation is unknown. Massive cardiomegaly occurs and death results at an early age from heart failure. Hers, H. G. a Glucosidase deficiency in generalized glycogen storage disease (Pompe's disease). Biochem. J. 86:11, 1963. Cori's Disease Also called type III glycogen storage disease, Cori's disease is caused by a deficiency of the glycogen debranching enzyme. Glycogen accumulates because only the outer branches can be removed from the molecule by phosphorylase. Hepatomegaly occurs but diminishes with age. The clinical manifestations are similar to but much milder than those seen in von Gierke's disease, because gluconeogenesis is unaffected, and hypoglycemia and its complications are less severe. Van Hoff, F., and Hers, H. G. The subgroups of type III glycogenesis. Eur. J. Biochem. 2:265, 1967. McArdle's Disease Also called the type V glycogen storage disease, McArdle's disease is caused by an absence of muscle phosphorylase. Patients suffer from painful muscle cramps and are unable to perform strenuous exercise, presumably because muscle glycogen stores are not available to the exercising muscle. Thus the normal increase in plasma lactate (released from the muscle) following exercise is absent. The muscles are probably damaged because of inadequate energy supply and glycogen accumulation. Release of muscle enzymes creatine kinase and aldolase and of myoglobin is common; elevated levels of these substances in the blood suggests a muscle disorder. McArdle, B. Myopathy due to a defect in muscle glycogen breakdown. Clin. Sci. 10:13, 1951. for glycogenolysis followed by glycolysis is Debranching Enzyme Is Required for Complete Hydrolysis of Glycogen The first enzyme involved in glycogen degradation, glycogen phosphorylase, is specific for a 1,4glycosidic linkages. However, it stops attacking a 1,4glucosidic linkages four glucosyl residues from an a 1,6branch point. A glycogen molecule that has been degraded by phosphorylase to the limit caused by the branches is called phosphorylaselimit dextrin. The action of a debranching Page 318 enzyme is what allows glycogen phosphorylase to continue to degrade glycogen. Debranching enzyme is a bifunctional enzyme that catalyzes two reactions necessary for debranching of glycogen. The first is a 4 a Dglucanotransferase activity in which a strand of three glucosyl residues is removed from a four glucosyl residue branch of the glycogen molecule (Figure 7.53). The strand remains covalently attached to the enzyme until it can be transferred to a free 4hydroxyl of a glucosyl residue at the end of the same or an adjacent glycogen molecule. The result is a longer amylose chain with only one glucosyl residue remaining in a 1,4linkage. This linkage is broken hydrolytically by the second enzyme activity of debranching enzyme, which is its amyloa 1,6glucosidase activity: The cooperative and repetitive action of phosphorylase and debranching enzyme results in complete phosphorolysis and hydrolysis of glycogen. Glycogen Figure 7.53 Action of the glycogen debranching enzyme. Page 319 storage diseases result when either of these enzymes is defective. The average molecule of glycogen yields about 12 molecules of glucose 1phosphate by action of phosphorylase for every molecule of free glucose produced by action of debranching enzyme. There is another, albeit quantitatively less important, pathway for glycogen degradation. A defect in this minor pathway, however, creates a major problem. As pointed out in Clin. Corr. 7.11, a glucosidase present in lysosomes degrades glycogen that enters these organelles during normal turnover of intracellular components. Synthesis of Glycogen Requires Unique Enzymes Figure 7.54 Pathway of glycogen synthesis. The first reaction involved in glycogen synthesis (Figure 7.54) is already familiar, being catalyzed by glucokinase in hepatic tissue and hexokinase in peripheral tissues: Phosphoglucomutase, discussed in relation to glycogen degradation, catalyzes a readily reversible reaction as follows: A unique reaction found at the next step involves formation of UDPglucose by action of glucose 1phosphate uridylyltransferase: This reaction generates UDPglucose, sometimes called "activated glucose" because of its large negative free energy of hydrolysis, which is used to build the glycogen molecule. Formation of UDPglucose is made energetically favorable and irreversible by hydrolysis of pyrophosphate by pyrophosphatase: Glycogen Synthase Glycogen synthase catalyzes transfer of the activated glucosyl moiety of UDPglucose to a glycogen molecule to form a new glycosidic bond between the hydroxyl group of C1 of the activated sugar and C4 of a glucosyl residue of the growing glycogen chain. The reducing end of glucose (C1 of glucose is an aldehyde that can reduce other compounds) is always added to a nonreducing end of the glycogen chain. The glycogen molecule, regardless of its size, theoretically has only one free reducing end tucked away within its core. UDP formed as a product of glycogen synthase is converted back to UTP by action of nucleoside diphosphate kinase: Glycogen synthase creates chains of glucose molecules with a 1,4glycosidic linkages, but does not form the a 1,6glycosidic branches found in glycogen. Its action alone would only produce a amylose, a straightchain polymer of glucose with a 1,4glycosidic linkages. Once an amylose chain of at least 11 residues has been formed, a "branching" enzyme comes into play. Its name is 1,4a glucan branching enzyme because it removes a block of about seven glucosyl residues from a growing chain and transfers it to another chain to produce an a 1,6 linkage (see Figure 7.55). The new branch has to be introduced at least four glucosyl residues from an adjacent branch point. Thus the creation of the highly branched structure of glycogen requires the concerted efforts of glycogen synthase and branching enzyme. The overall balanced equation for Page 320 Figure 7.55 Action of the glycogen branching enzyme. glycogen synthesis by the pathway just outlined is As noted above, the combination of glycogenolysis and glycolysis yields only three molecules of ATP per glucosyl residue: Thus the combination of glycogen synthesis plus glycogen degradation to lactate actually yields only one ATP. However, glycogen synthesis and degradation are normally carried out at different times in a cell. For example, white muscle fibers synthesize glycogen at rest when glucose is plentiful and less ATP is needed for muscle contraction. Glycogen is then used during periods of exertion. Although in such terms glycogen storage is not a very efficient process, it provides cells with a fuel reserve that can be very quickly and efficiently mobilized. Page 321 Special Features of Glycogen Degradation and Synthesis Why Store Glucose As Glycogen? Since glycogen is such a good fuel reserve, it is obvious why we synthesize and store glycogen in liver and muscle. But why not store our excess glucose calories entirely as fat instead of glycogen? The answer is at least threefold: (1) we do store fat, but fat cannot be mobilized nearly as rapidly in muscle as glycogen; (2) fat cannot be used as a source of energy in the absence of oxygen; and (3) fat cannot be converted to glucose to maintain blood glucose levels. Why not just pump glucose into cells and store it as free glucose until needed? Why waste so much ATP making a polymer out of glucose? The problem is that glucose is osmotically active. It would cost ATP to "pump" glucose into a cell against a concentration gradient, and glucose would have to reach concentrations of 400 mM in liver cells to match the "glucose reserve" provided by the usual liver glycogen levels. Unless balanced by outward movement of some other osmotically active compound, accumulation of such concentrations of glucose would cause uptake of considerable water and osmotic lysis of the cell. Assuming the molecular mass of a glycogen molecule is of the order of 107 Da, 400 mM glucose is in effect stored at an intracellular glycogen concentration of 0.01 mM. Storage of glucose as glycogen therefore creates no osmotic pressure problem for the cell. Glycogenin Is Required As a Primer for Glycogen Synthesis Like DNA synthesis, a primer is needed for glycogen synthesis. No template, however, is required. Glycogen itself is the usual primer, in that glycogen synthesis can take place by addition of glucosyl units to glycogen "core" molecules, which are almost invariably present in the cell. The outer regions of the glycogen molecule get removed and resynthesized more rapidly than the inner core. Glycogen within a cell is frequently sheared by the combined actions of glycogen phosphorylase and debranching enzyme but is seldom obliterated before glycogen synthase and branching enzyme rebuild the molecule. This begs the question why glycogen is a branched molecule with only one real beginning (the reducing end) and many branches terminating with nonreducing glucosyl units. The answer is that this gives numerous sites of attack for glycogen phosphorylase on a mature glycogen molecule and the same number of sites that function as primers for the addition of glucosyl units by glycogen synthase. If cells synthesized a amylose, that is, an unbranched glucose polymer, there would only be one nonreducing end per molecule. This would surely make glycogen degradation and synthesis much slower. As it is, glycogen phosphorylase and glycogen synthase are found in tight association with glycogen granules in a cell. By taking up residence in the branches of the glycogen tree, both enzymes have ready access to a multitude of nonreducing sugars at the ends of the limbs. Figure 7.56 Glycogenin provides a primer for glycogen synthesis by glycogen synthase. Tyr designates a tyrosine residue of glycogenin. But why is a primer needed for glycogen synthesis? It turns out to be impossible to initiate glycogen synthesis with simply a glucose molecule as the acceptor of an activated glucosyl residue from UDPglucose. Glycogen synthase has a very low Km for very large glycogen molecules and therefore readily adds glucosyl residues to make even larger glycogen molecules. However, the Km gets larger and larger as the glycogen molecule gets smaller and smaller. This phenomenon is so pronounced that glucose, at its physiological concentration, could never function as a primer. This led for some time to the notion that glycogen must be immortal; that is, some glycogen must be handed down from one cell generation to the next in order for glycogen to be synthesized. However, it is now known that a polypeptide of 332 amino acids called glycogenin functions as a primer for glycogen synthesis. Glycogenin is a selfglucosylating enzyme that uses UDPglucose to link glucose to one of its own tyrosine residues (Figure 7.56). Glycosylated glycogenin then serves as a primer for synthesis of glycogen. Alas, glycogen is not immortal. Page 322 Glycogen Limits Its Own Synthesis If glycogen synthase becomes more efficient as the glycogen molecule gets bigger, how is synthesis of this ball of sugar curtailed? Fat cells have an almost unlimited capacity to pack away fat— but then fat cells have nothing else to do. Muscle cells participate in mechanical activity and liver cells carry out many processes other than glycogen synthesis. Even in the face of excess glucose, there has to be a way to limit the intracellular accumulation of glycogen. Glycogen itself inhibits glycogen synthase by a mechanism discussed later (see p. 326). Glycogen Synthesis and Degradation Are Highly Regulated Pathways Glycogen synthase and glycogen phosphorylase are regulatory enzymes of glycogen synthesis and degradation, respectively. Both catalyze nonequilibrium reactions, and both are subject to control by allosteric effectors and covalent modification. Regulation of Glycogen Phosphorylase Glycogen phosphorylase is subject to allosteric activation by AMP and allosteric inhibition by glucose and ATP (Figure 7.57). Control by these effectors is integrated with a very elaborate control by covalent modification. Phosphorylase exists in an a form, which is active, and a b form, which is inactive. These forms are interconverted by the actions of phosphorylase kinase and phosphoprotein phosphatase (Figure 7.57). A conformational change caused by phosphorylation transforms the enzyme into a more active catalytic state. Phosphorylase b has some catalytic activity and can be greatly activated by AMP. This allosteric effector has little activating effect, however, on the already active phosphorylase a. Hence the covalent modification mechanism can be bypassed by the allosteric mechanism and vice versa. Phosphorylase kinase is responsible for phosphorylation and activation of phosphorylase (Figure 7.57). Moreover, phosphorylase kinase itself is also sub Figure 7.57 Regulation of glycogen phosphorylase by covalent modification. Phosphorylation converts glycogen phosphorylase and phosphorylase kinase from their inactive b forms to their active a forms. Page 323 ject to regulation by a cyclic phosphorylation–dephosphorylation mechanism. Protein kinase A phosphorylates and activates phosphorylase kinase; phosphoprotein phosphatase in turn dephosphorylates and inactivates phosphorylase kinase. Phosphorylase kinase is a large enzyme complex (1.3 × 106 Da), composed of four subunits with four molecules of each subunit in the complex (a 4b 4g4d 4). Catalytic activity resides with the g subunit; a , b , and d subunits exert regulatory control. The a and b subunits are phosphorylated in the transition from the inactive b form to the active a form of the enzyme. Protein kinase A can only exert an effect on phosphorylase via its ability to phosphorylate and activate phosphorylase kinase. Thus a bicyclic system is required for activation of phosphorylase in response to cAMPmediated signals. The d subunit of phosphorylase kinase also plays a regulatory role. It corresponds to a Ca2+binding regulatory protein, called calmodulin. Not unique to phosphorylase kinase, calmodulin is found in cells as the free molecule and also bound to other enzyme complexes. It functions as a Ca2+ receptor in the cell, responding to changes in intracellular Ca2+ concentration and affecting the relative activities of a number of enzyme systems. Binding of Ca2+ to the calmodulin subunit of phosphorylase kinase changes the conformation of the complex, making the enzyme more active with respect to the phosphorylation of phosphorylase. As shown in Figure 7.57, Ca2+ is an activator of both phosphorylase kinase a and phosphorylase kinase b. Maximum activation of phosphorylase kinase requires both phosphorylation of specific serine residues of the enzyme and interaction of Ca2+ with the calmodulin subunit of the enzyme. This is one mechanism by which Ca2+ functions as an important ''second messenger" of hormone action, as will be discussed below. Activation of phosphorylase kinase by phosphorylation and Ca2+ will have a substantial effect on the activity of glycogen phosphorylase. It is equally obvious, however, that turning off the phosphoprotein phosphatase that modulates the phosphorylation states of both phosphorylase kinase and glycogen phosphorylase (Figure 7.57) could achieve the same effect. Ultimate control of glycogen phosphorylase would involve the reciprocal regulation of phosphoprotein phosphatase and phosphorylase kinase activities. Although numerous details remain to be understood, there is evidence that activities of phosphoprotein phosphatase and phosphorylase kinase are controlled in a reciprocal manner. Regulation of phosphoprotein phosphatase activity is linked to cAMP (see p. 325). The important point in Figure 7.57 is that hormones that increase cAMP levels, such as glucagon and epinephrine, promote activation of glycogen phosphorylase by signaling activation of phosphorylase kinase and inactivation of phosphoprotein phosphatase. On the other hand, insulin, which acts either though a second messenger or a kinasemediated signal cascade (see p. 879), exerts the opposite effect on phosphorylase by promoting activation of phosphoprotein phosphatase activity. The Cascade that Regulates Glycogen Phosphorylase Amplifies a Small Signal into a Very Large Effect There is a good reason for the existence of the bicyclic control system for phosphorylation of glycogen phosphorylase. It provides a tremendous amplification mechanism of a very small initial signal. Activation of adenylate cyclase by one molecule of epinephrine causes formation of many molecules of cAMP. Each cAMP molecule activates a protein kinase A molecule, which in turn activates many molecules of phosphorylase kinase as well as many molecules of phosphoprotein phosphatase. In turn, phosphorylase kinase phosphorylates many molecules of glycogen phosphorylase, which in turn catalyze phosphorolysis of many glycosidic bonds of glycogen. A very elaborate amplification system is therefore provided in which the signal provided by just a few molecules of hormone is amplified into production of an enormous number of glucose 1phosphate molecules. If each step represents, for argument's sake, an amplification factor of 100, then a total of four steps would result in an amplification Page 324 of 100 million! This system is so rapid, in large part because of this amplification mechanism, that all of the stored glycogen of white muscle fibers could be completely mobilized within just a few seconds. Regulation of Glycogen Synthase Glycogen synthase has to be active for glycogen synthesis and inactive for glycogen degradation. The combination of the reactions catalyzed by glycogen synthase, glycogen phosphorylase, glucose 1phosphate uridylyltransferase, and nucleoside diphosphate kinase adds up to a futile cycle with the overall equation ATP ADP + Pi. Hence glycogen synthase needs to be turned off when glycogen phosphorylase is turned on, and vice versa. Activation of glycogen synthase by glucose 6phosphate, an allosteric effector, is probably of physiological significance under some circumstances (Figure 7.58). However, as with glycogen phosphorylase, this mode of control is integrated with regulation by covalent modification (Figure 7.58). Glycogen synthase exists in two forms. One is designated the D form because it is dependent on the presence of G6P for activity. The other is designated the I form because its activity is independent of the presence of G6P. The D form corresponds to the b or inactive form of the enzyme, the I form to the a or active form of the enzyme. Phosphorylation of glycogen synthase is catalyzed by several different kinases, which in turn are regulated by second messengers of Figure 7.58 Regulation of glycogen synthase by covalent modification. Phosphorylation converts glycogen synthase from its active a form to its inactive b form. Page 325 2+ hormone action, including cAMP, Ca , diacylglycerol, and probably yet to be identified compounds. Each of the protein kinases shown in Figure 7.58 is capable of catalyzing the phosphorylation and contributing to inactivation of glycogen synthase. Although glycogen synthase is a simple tetramer (a 4) of only one subunit type (mol wt 85,000 Da), it can be phosphorylated on at least nine different serine residues. Eleven different protein kinases have been identified that can phosphorylate glycogen synthase. This stands in striking contrast to glycogen phosphorylase, which is regulated by phosphorylation of one site by one specific kinase. Cyclic AMP is an extremely important intracellular signal for reciprocally controlling glycogen synthase (Figure 7.58) and glycogen phosphorylase (Figure 7.57). An increase in cAMP signals activation of glycogen phosphorylase and inactivation of glycogen synthase via activation of protein kinase A and inhibition of phosphoprotein phosphatase. Ca2+ likewise can influence the phosphorylation state of both enzymes and reciprocally regulate their activity via its effects on phosphorylase kinase. Two cAMPindependent, Ca2+activated protein kinases have been identified that also may have physiological significance. One of these is a calmodulindependent protein kinase and the other a Ca2+and phospholipiddependent protein kinase (protein kinase C). Both enzymes phosphorylate glycogen synthase, but neither can phosphorylate glycogen phosphorylase. Protein kinase C requires phospholipid, diacylglycerol, and Ca2+ for full activity. There is considerable interest in protein kinase C because tumorpromoting agents called phorbol esters have been found to mimic diacylglycerol as activators of this enzyme. Diacylglycerol is considered an important "second messenger" of hormone action, acting via protein kinase C to regulate numerous cellular processes (see p. 865). Glycogen synthase is also phosphorylated by glycogen synthase kinase3, casein kinase I, and casein kinase II. These kinases are not subject to regulation by cAMP or Ca2+. It is likely, however, that special regulatory mechanisms exist to regulate these kinases. Herein may lie solutions to unsolved problems such as the mechanism of action of insulin and other hormones. The phosphoprotein phosphatase that converts glycogen synthase b back to glycogen synthase a (Figure 7.58) is regulated in a manner analogous to that described in the discussion of glycogen phosphorylase regulation (Figure 7.57). Cyclic AMP promotes inactivation whereas insulin promotes activation of glycogen synthase through opposite effects on phosphoprotein phosphatase activity. Regulation of Phosphoprotein Phosphatases Figure 7.59 Mechanism for regulation of a phosphatase that binds to glycogen. The glycogenbinding subunit G binds directly to glycogen; the phosphoprotein phosphatase catalytic subunit C binds to glycogen via the G subunit; and the phosphorylated inhibitor 1 (I1) binds the free catalytic subunit. About a dozen different phosphoprotein phosphatases with specificity for removal of phosphate from serine residues of proteins are currently being studied. In general, phosphoprotein phosphatases occur as catalytic subunits associated with a number of different regulatory subunits that control the activity of the catalytic subunit, determine which substrate(s) the catalytic subunit can interact with and dephosphorylate, and target the association of a catalytic subunit with a specific structure or component within a cell. One such regulatory protein important for glycogen metabolism has been given the name G subunit, denoting a glycogenbinding protein. G subunit binds both glycogen and a phosphatase catalytic subunit (Figure 7.59). This association makes the phosphatase ten times more active toward glycogen synthase and glycogen phosphorylase and thereby greatly promotes their dephosphorylation. However, phosphorylation of the G subunit by protein kinase A results in release of the phosphatase catalytic subunit, which is then less active. Interaction of the free catalytic subunit with yet another regulatory protein (called inhibitor 1) then causes further inhibition of phosphatase activity. Effective inhibition of the residual phosphatase activity of the catalytic subunit requires phosphorylation of inhibitor 1 by protein kinase A, thereby creating yet another link to hormones that increase cAMP levels. Insulin has effects opposite to those of cAMP; Page 326 that is, insulin promotes activation of the catalytic subunit of phosphoprotein phosphatase. This presumably involves reversal of the steps promoted by cAMP, but details of how this is accomplished remain to be established. Effector Control of Glycogen Metabolism Certain muscles are known to mobilize their glycogen stores rapidly in response to anaerobic conditions without marked conversion of phosphorylase b to phosphorylase a or glycogen synthase a to glycogen synthase b. Presumably this is accomplished by effector control in which ATP levels decrease, causing less inhibition of phosphorylase; glucose 6phosphate levels decrease, causing less activation of glycogen synthase; and AMP levels increase, causing activation of phosphorylase. This enables muscle to keep working, for at least a short period of time, by using ATP produced by glycolysis of glucose 6phosphate obtained from glycogen. Proof that effector control can operate has also been obtained in studies of a special strain of mice that are deficient in muscle phosphorylase kinase. Phosphorylase b in muscle of such mice cannot be converted into phosphorylase a. Nevertheless, heavy exercise of these mice results in depletion of muscle glycogen, presumably because of stimulation of phosphorylase b by effectors. Negative Feedback Control of Glycogen Synthesis by Glycogen Glycogen exerts feedback control over its own formation. The portion of glycogen synthase in the active a form decreases as glycogen accumulates in a particular tissue. The mechanism is not well understood, but glycogen may make the a form a better substrate for one of the protein kinases, or, alternatively, glycogen may inhibit dephosphorylation of glycogen synthase b by phosphoprotein phosphatase. Either mechanism would account for the shift in the steady state in favor of glycogen synthase b that occurs in response to glycogen accumulation. Phosphorylase a Functions As a "Glucose Receptor" in the Liver Consumption of a carbohydratecontaining meal results in an increase in blood and liver glucose, which signals an increase in glycogen synthesis in the latter tissue. The mechanism involves glucose stimulation of insulin release from the pancreas and its effects on hepatic glycogen phosphorylase and glycogen synthase. However, hormoneindependent mechanisms also appear to be important in liver (Figure 7.60). Direct inhibition of phosphorylase a by glucose is probably of importance. Binding of glucose to phosphorylase makes the a form of phosphorylase a better substrate for dephosphorylation by phosphoprotein phosphatase. Therefore phosphorylase a functions as a glucose receptor in liver. Binding of glucose to phosphorylase a promotes inactivation of phosphorylase a, with the overall result being inhibition of glycogen degradation by glucose. This "negative feedback" control of glycogenolysis by glucose would not necessarily promote glycogen synthesis. However, there also is evidence that phosphorylase a is an inhibitor of the dephosphorylation of glycogen synthase b by phosphoprotein phosphatase. This inhibition is lost once phosphorylase a has been converted to phosphorylase b (Figure 7.60). In other words, phosphoprotein phosphatase can turn its attention to glycogen synthase b only following dephosphorylation of phosphorylase a. Thus, as a result of interaction of glucose with phosphorylase a, phosphorylase becomes inactivated, glycogen synthase becomes activated, and glycogen is synthesized rather than degraded in liver. Phosphorylase a can serve this function of "glucose receptor" in liver because the concentration of glucose in liver always reflects the blood concentration of glucose. This is not true for extrahepatic tissues. Liver cells have a very high capacity transport system for glucose and a high Km enzyme for glucose phosphorylation (glucokinase). Cells of extrahepatic Page 327 Figure 7.60 Overview of the mechanism responsible for glucose stimulation of glycogen synthesis in the liver. tissues as a general rule have glucose transport and phosphorylation systems that maintain intracellular glucose at concentrations too low for phosphorylase a to function as a "glucose receptor." Glucagon Stimulates Glycogen Degradation in the Liver Glucagon is released from a cells of pancreas in response to low blood glucose levels. One of glucagon's primary jobs during periods of low food intake (fasting or starvation) is to mobilize liver glycogen, that is, stimulate glycogenolysis, in order to ensure that adequate blood glucose is available to meet the needs of glucose dependent tissues. Glucagon circulates in blood until it interacts with glucagon receptors such as those located on the plasma membrane of liver cells (see Figure 7.61). Binding of glucagon to these receptors activates adenylate cyclase and triggers the cascades that result in activation of glycogen phosphorylase and inactivation of glycogen synthase by the mechanisms given in Figures 7.57 and 7.58, respectively. Glucagon also inhibits glycolysis at the level of 6phosphofructo1kinase and pyruvate kinase by the mechanisms given in Figures 7.25 and 7.30, respectively. The net result of these effects of glucagon, all mediated by the second messenger cAMP and covalent modification, is a very rapid increase in blood glucose levels. Hyperglycemia might be expected but does not occur because less glucagon is released from the pancreas as blood glucose levels increase. Epinephrine Stimulates Glycogen Degradation in the Liver Epinephrine is released into blood from chromaffin cells of the adrenal medulla in response to stress. This hormone is our "fright, flight, or fight" hormone, preparing the body for either combat or escape. Figure 7.61 Cyclic AMP mediates the stimulation of glycogenolysis in liver by glucagon and b agonists (epinephrine). See legends for Figures 7.19 and 7.25. Epinephrine interacts directly with receptors in the plasma membrane of liver cells to activate adenylate cyclase (Figure 7.61). The resulting increase in cAMP has the same effect as that caused by glucagon, that is, activation of glycogenolysis and inhibition of glycogenesis and glycolysis to maximize the release of glucose from liver. The plasma membrane receptor for epinephrine, Page 328 which is in communication with adenylate cyclase, is the b adrenergic receptor. The plasma membrane of liver cells also has another binding protein for epinephrine, called the a adrenergic receptor. Interaction of epinephrine with a adrenergic receptors leads to formation of inositol 1,4,5trisphosphate (IP3) and diacylglycerol (Figure 7.62). These compounds are second messengers, produced in the plasma membrane by the action of a phospholipase C on phosphatidylinositol 4,5bisphosphate (Figure 7.63). Inositol 1,4,5trisphosphate stimulates the release of Ca2+ from the endoplasmic reticulum (Figure 7.62). As previously discussed (Figure 7.57), the increase in Ca2+ activates phosphorylase kinase, which in turn activates glycogen phosphorylase. Likewise (Figure 7.58), Ca2+ mediated activation of phosphorylase kinase, calmodulindependent protein kinase, and protein kinase C, as well as diacylglycerolmediated activation of protein kinase C, may all be important for inactivation of glycogen synthase. Figure 7.62 Inositol trisphosphate (IP3) and Ca2+ mediate the stimulation of glycogenolysis in liver by a agonists. The adrenergic receptor and glucose transporter are intrinsic components of the plasma membrane. Although not indicated, phosphatidylinositol 4,5bisphosphate (PIP2) is also a component of the plasma membrane. The consequences of epinephrine action is an increased release of glucose into the blood from the glycogen stored in liver. This makes more blood glucose available to tissues that are called upon to meet the challenge of the stressful situation that triggered the release of epinephrine from adrenal medulla. Epinephrine Stimulates Glycogen Degradation in Heart and Skeletal Muscle Epinephrine also stimulates glycogen degradation in heart and skeletal muscle. Cyclic AMP, produced in response to epinephrine stimulation of adenylate cyclase via b adrenergic receptors (Figure 7.64), signals concurrent activation of glycogen phosphorylase and inactivation of glycogen synthase by mechanisms given previously in Figures 7.57 and 7.58, respectively. This does not lead, however, to glucose release into blood from these tissues. In contrast to liver, heart and skeletal muscle lack glucose 6phosphatase, and in these tissues cAMP does not inhibit but rather stimulates glycolysis (see Figure 7.28). Thus the role of epinephrine on glycogen metabolism in heart and skeletal muscle is to make more glucose 6phosphate available for glycolysis. ATP generated by glycolysis can then be used to meet the metabolic demand imposed on these muscles by the stress that triggered epinephrine release. Neural Control of Glycogen Degradation in Skeletal Muscle Nervous excitation of muscle activity is mediated via changes in intracellular Ca2+ concentrations (Figure 7.65). A nerve impulse causes membrane depo Figure 7.63 Phospholipase C cleaves phosphatidylinositol 4,5bisphosphate to produce 1,2diacylglycerol and inositol 1,4,5trisphosphate. Page 329 Figure 7.64 Cyclic AMP mediates the stimulation of glycogenolysis in muscle by b agonists (epinephrine). The adrenergic receptor is an intrinsic component of the plasma membrane that acts to stimulate adenylate cyclase via a stimulatory Gprotein (Gs). Figure 7.65 Ca2+ mediates the stimulation of glycogenolysis in muscle by nervous excitation. larization, which in turn causes Ca2+ release from the sarcoplasmic reticulum into the sarcoplasm of muscle cells. This release of Ca2+ triggers muscle contraction, whereas reaccumulation of Ca2+ by the sarcoplasmic reticulum causes relaxation. The same change in Ca2+ concentration effective in causing muscle contraction (from 10–8 to 10–6 M) also greatly affects the activity of phosphorylase kinase. As Ca2+ concentrations increase there is more muscle activity and a greater need for ATP. Activation of phosphorylase kinase by Ca2+ leads to the subsequent activation of glycogen phosphorylase and perhaps the inactivation of glycogen synthase. The result is that more glycogen is converted to glucose 6phosphate so that more ATP can be produced to meet the greater energy demand of muscle contraction. Insulin Stimulates Glycogen Synthesis in Muscle and Liver An increase in blood glucose signals release of insulin from b cells of the pancreas. Insulin circulates in blood, serving as a first messenger to inform several tissues that excess glucose is present. Insulin receptors, located on the plasma membranes of insulinresponsive cells, respond to insulin binding by either producing a second messenger of insulin action or inducing a protein kinase cascade that promotes glucose use within these tissues (Figures 7.66 and 7.67). The pancreas responds to a decrease in blood glucose with less release of insulin but greater release of glucagon. These hormones have opposite effects on glucose utilization by liver, thereby establishing the pancreas as a finetuning device that prevents dangerous fluctuations in blood glucose levels.