LEHNINGER PRINCIPLES OF BIOCHEMISTRY - VOL. 2. BIOENERGETICS AND METABOLISM - 2014

PART II. BIOENERGETICS AND METABOLISM

15. PRINCIPLES OF METABOLIC REGULATION

15.5. Coordinated Regulation of Glycogen Synthesis and Breakdown

As we discussed above, the mobilization of stored Glycogen begins with the action of Glycogen phosphorylase, which cleaves glucose-1-phosphate molecules from glycogen (Fig. 15-25). The Regulation of Glycogen phosphorylase is a classic and highly instructive example of enzymatic control. It was one of the first Enzymes found to be subject to Allosteric Regulation and the first for which activity was shown to be controlled by reversible phosphorylation. Furthermore, it was among the first allosteric Proteins whose detailed three-dimensional structures in both active and inactive states were determined by X-ray crystallography. Glycogen phosphorylase also serves as a prime example of the diverse Functions of isozymes in various Tissues.

Glycogen Phosphorylase Is Subject to Allosteric and Hormonal Regulation

In the late 1930s, Carl and Gerty Cori (Box 15-4) discovered two interconvertible forms of Muscle glycogen phosphorylase: the catalytically active glycogen phosphorylase a and the less active glycogen phosphorylase b (Fig. 15-34). Later studies by Earl Sutherland revealed that resting muscle contains predominantly phosphorylase b, whereas during active Muscle contraction, the hormone epinephrine triggers the phosphorylation of a specific Ser residue in phosphorylase b, converting it into the more active phosphorylase a. Note that glycogen phosphorylase is frequently referred to simply as phosphorylase, as it was the first phosphorylase to be studied; this shortened name has become standard in the literature.

Class="center">Earl Sutherland Jr., 1915–1974

Fig. 15-34. Regulation of Muscle glycogen phosphorylase by covalent modification. In the more active form of the enzyme, termed phosphorylase a, the Ser¹⁴ residue in each subunit is phosphorylated. Phosphorylase a is converted to the less active phosphorylase b by the enzymatic removal of these phosphoryl groups, catalyzed by phosphorylase a phosphatase (PP1). Phosphorylase b can be converted back to active phosphorylase a by phosphorylase b kinase. (For The regulation of glycogen phosphorylase, see also Fig. 6-36.)

The enzyme responsible for activating phosphorylase via The transfer of a phosphoryl group to a Ser residue—phosphorylase b kinase—is itself activated by epinephrine or Glucagon through a cascade of reactions shown in Figure 15-35. Sutherland discovered the second messenger cAMP, whose concentration increases upon stimulation by epinephrine (in muscle) or glucagon (in Liver). Elevated cAMP levels initiate an enzyme cascade—in which one catalyst activates a second, which in turn activates a third. This cascade mechanism greatly amplifies the initial signal (see the pink-shaded area in Fig. 15-35). An increase in cAMP concentration activates cAMP-dependent protein kinase, also known as protein kinase A (PKA). PKA then phosphorylates and activates phosphorylase b kinase, which catalyzes the phosphorylation of a Ser residue in each of the two identical subunits of glycogen phosphorylase, thereby activating the enzyme and stimulating glycogen breakdown. In muscle, this process supplies the energy for Glycolysis needed to sustain the "fight-or-flight" response signaled by epinephrine. In the liver, glycogen breakdown and glucose production counteract falling Blood glucose levels, with glucagon serving as the signal. The distinct physiological roles of epinephrine and glucagon are reflected in the subtle regulatory differences between muscle and liver enzymes. Muscle and liver glycogen phosphorylases are isozymes encoded by different genes and feature distinct regulatory properties.

Fig. 15-35. The cascade mechanism of epinephrine and glucagon action. The binding of epinephrine to muscle Cells (left) or glucagon to liver cells (right), mediated by specific Cell-surface receptors, leads to the Activation of a GTP-binding protein (see Fig. 12-12). Active G_sα stimulates an increase in cAMP concentration, thereby activating PKA. These events trigger a series of phosphorylation reactions; PKA activates phosphorylase b kinase, which subsequently activates glycogen phosphorylase. This cascade mechanism results in a massive Amplification of the initial signal; numbers in the pink boxes indicate the lower Structure/21.html">Limits of the actual increase in molecular numbers at each step of the cascade. Glycogen breakdown yields glucose, which in myocytes can serve as an ATP source (via glycolysis) for muscle activity, or be released into the bloodstream by hepatocytes to correct abnormally low blood glucose levels.

In muscle, phosphorylase regulation involves both covalent modification and two allosteric mechanisms (Fig. 15-35). Ca²⁺ ions, which signal muscle contraction, bind to phosphorylase b kinase and activate it, converting it into the active a form. Ca²⁺ ions bind to phosphorylase b kinase via its δ subunit, which is calmodulin (see Fig. 12-11). During intense muscle contraction, ATP breakdown leads to the accumulation of AMP, which binds to phosphorylase and allosterically activates it, thereby accelerating the release of glucose-1-phosphate from glycogen. Conversely, high concentrations of ATP bind to the allosteric site normally occupied by AMP, thereby inhibiting phosphorylase.

When muscle returns to the resting state, the enzyme phosphorylase a phosphatase, also known as phosphoprotein phosphatase-1 (PP1), removes the phosphate group from phosphorylase a, converting it back to the less active phosphorylase b.

Like the muscle enzyme, liver glycogen phosphorylase is subject to both hormonal control (via phosphorylation and dephosphorylation) and allosteric regulation. The dephosphorylated form of the enzyme is virtually inactive. During periods of very low blood glucose, glucagon (acting through the same cascade mechanism shown in Fig. 15-35) activates phosphorylase b kinase, converting phosphorylase to the active a form and promoting glucose release into the blood. When blood glucose levels return to normal, glucose enters hepatocytes and binds to an inhibitory allosteric site on the phosphorylase a molecule. This binding induces a conformational change that exposes the phosphorylated Ser residues to PP1, which catalyzes their dephosphorylation and inactivates phosphorylase (Fig. 15-36). The presence of this allosteric glucose-binding site enables liver glycogen phosphorylase to function as a glucose sensor, responding appropriately to fluctuations in blood glucose levels.

Fig. 15-36. Liver glycogen phosphorylase as a glucose sensor. Glucose binding to the allosteric site of the liver phosphorylase a isozyme induces a conformational change that exposes the phosphorylated Ser residues to phosphorylase a phosphatase (PP1). This phosphatase converts phosphorylase a to phosphorylase b, sharply reducing enzyme activity and slowing glycogen breakdown when blood glucose levels are high. Insulin also indirectly stimulates PP1 and suppresses Glycogenolysis.

Glycogen synthase is Also Regulated by Phosphorylation-Dephosphorylation

Like glycogen phosphorylase, glycogen synthase exists in phosphorylated and dephosphorylated forms (Fig. 15-37). The active form of this enzyme—glycogen synthase a—is unphosphorylated. Phosphorylation of hydroxyl groups on specific Ser residues in both enzyme subunits converts glycogen synthase a into glycogen synthase b, which is active only in the presence of its allosteric activator, glucose-6-phosphate. Interestingly, phosphorylation of various residues on glycogen synthase can be carried out by as many as 11 different protein Kinases. The most prominent role in regulation is played by glycogen synthase kinase 3 (GSK3), which attaches phosphate groups to three Serine residues near the C-terminus of the glycogen synthase molecule, leading to its complete inactivation. The action of GSK3 is hierarchical: it cannot phosphorylate glycogen synthase unless a nearby residue has already been phosphorylated (primed) by casein kinase II (CKII; Fig. 15-38a).

Fig. 15-37. Regulation of glycogen synthase activity by GSK3. The active enzyme form, glycogen synthase a, contains three Ser residues near its C-terminus that are phosphorylated by glycogen synthase kinase 3 (GSK3), converting the enzyme into the inactive b form (GSb). GSK3 can act only after casein kinase II (CKII) has phosphorylated a neighboring residue. Insulin promotes the activation of glycogen synthase b by inhibiting GSK3 (see Fig. 12-16 for details) and stimulating phosphoprotein phosphatase (PP1 in muscle and another phosphatase in liver). In muscle, epinephrine activates PKA, which phosphorylates the glycogen-targeting subunit G_M (see Fig. 15-40) at a specific site, causing the release of PP1 from the glycogen-protein complex. Glucose-6-phosphate favors the dephosphorylation of glycogen synthase because its binding induces a conformation that makes the enzyme a better substrate for PP1. Glucose also promotes dephosphorylation; its binding to glycogen phosphorylase a triggers a conformational change that facilitates its dephosphorylation and conversion to glycogen phosphorylase b, thereby relieving the inhibition of PP1 (see Fig. 15-39).

Fig. 15-38. Phosphorylation of glycogen synthase by GSK3. (a) Glycogen synthase kinase 3 first binds to its substrate (glycogen synthase) via Electrostatic Interactions between three positively charged residues (Arg⁹⁶, Arg¹⁸⁰, and Lys²⁰⁵) and a phosphorylated Ser residue at the +4 position of the substrate. (Here, the Thr or Ser residue in the substrate that is phosphorylated first is designated position 0. Residues closer to the N-terminus are numbered -1, -3, etc., and those closer to the C-terminus are designated +1, +2, etc.) This interaction aligns the Active Site of the enzyme with the Ser residue at position 0, which is then phosphorylated. Afterward, the enzyme shifts along the polypeptide chain and sequentially phosphorylates Ser residues at positions -4 and -8. (b) The N-terminal region of GSK3 contains a Ser residue that can be phosphorylated by PKA or PKB (see Fig. 15-39). This phosphorylation creates a pseudosubstrate motif that blocks the active site and prevents access to the normal substrate. This inhibits GSK3 activity until the phosphate group in this pseudosubstrate region is removed by PP1. For GSK3 to initiate its action, other substrate proteins must also be pre-phosphorylated at the +4 position by another protein kinase. (For the regulation of glycogen synthase, see also Figs. 6-37 and 12-22b.)

In the liver, The conversion of glycogen synthase b into its active form is mediated by PP1, which is bound to the glycogen particle. PP1 removes the phosphate groups from three serine residues that had been phosphorylated by GSK3. Glucose-6-phosphate can bind to an allosteric site on the glycogen synthase b molecule, thereby enhancing the dephosphorylating activity of PP1. By analogy with glycogen phosphorylase, which functions as a glucose sensor, glycogen synthase can be viewed as a glucose-6-phosphate sensor. The role that PP1 plays in the liver can be fulfilled by another phosphatase in Muscles, which similarly activates glycogen synthase via dephosphorylation.

The Action of Insulin is mediated by glycogen synthase kinase 3

As we discussed in Chapter 12, one of the mechanisms of insulin action is the activation of protein kinase B (PKB), which in turn phosphorylates and inactivates GSK3 (Fig. 15-39; see also Fig. 12-16). Phosphorylation of the Ser residue in the N-terminal region of GSK3 converts this segment of the protein into a pseudosubstrate that occupies the site where the prephosphorylated serine residue would normally bind (Fig. 15-38, b). This prevents GSK3 from binding its normal phosphorylated substrate, thereby inactivating the enzyme and shifting the equilibrium toward the dephosphorylation of glycogen synthase by PP1. Glycogen phosphorylase can also influence the phosphorylation of glycogen synthase: active glycogen phosphorylase inhibits PP1, preventing it from activating glycogen synthase (Fig. 15-37).

Fig. 15-39. Interconnection between insulin, GSK3, and glycogen synthase. The binding of insulin to its receptor activates the receptor's Tyrosine kinase activity, leading to the phosphorylation of Insulin Receptor substrate 1 (IRS-1). The phosphotyrosine of this protein then binds phosphatidylinositol 3-kinase (PI3K), which converts phosphatidylinositol 4,5-bisphosphate (PIP₂) in The Plasma Membrane into phosphatidylinositol 3,4,5-trisphosphate (PIP₃). PDK-1, activated upon binding to PIP₃, activates a second protein kinase (PKB), which results in the phosphorylation of glycogen synthase kinase 3 (GSK3) and The formation of a "pseudosubstrate" domain within its molecule; this inactivates the enzyme According to the mechanism depicted in Fig. 15-38, b. The inactivation of GSK3 allows phosphoprotein phosphatase 1 (PP1) to dephosphorylate Glycogen synthase and convert it into the active state. Thus, insulin stimulates glycogen synthesis. The Mechanism of insulin action is illustrated in more detail in Fig. 12-16.

Although The Role of GSK3 in Glycogen METABOLISM was elucidated first (hence the enzyme's name, glycogen synthase kinase), it is clear that the enzyme has a much broader range of functions than merely regulating glycogen synthase. It mediates signal Transduction from insulin, other growth factors, and nutrients, and also participates in determining cell fate during embryonic development. Its targets include cytoskeletal proteins as well as key proteins involved in mRNA and Protein Synthesis. Like glycogen synthase, these targets must be phosphorylated by another protein kinase before GSK3 can act upon them.

The Central Role of phosphoprotein phosphatase 1 in glycogen metabolism

The same PP1 enzyme is capable of removing phosphate groups from all three enzymes that are phosphorylated in response to a glucagon signal (in the liver) and an epinephrine signal (in The Liver and muscles): phosphorylase kinase, glycogen phosphorylase, and glycogen synthase. Insulin stimulates glycogen synthesis by activating PP1 and inactivating GSK3.

PP1 does not exist in a free form in the Cytosol; it is tightly anchored to its target proteins (phosphorylase kinase, glycogen phosphorylase, and glycogen synthase) by one of the members of the glycogen-targeting protein family, which binds glycogen (Fig. 15-40). PP1 itself is subject to covalent and allosteric regulation; it is inactivated by PKA-catalyzed phosphorylation and activated through allosteric interaction with glucose-6-phosphate.

Fig. 15-40. The glycogen-targeting protein G_M. The glycogen-targeting protein belongs to a family of proteins that tether other proteins (including PP1) to glycogen granules. In response to an insulin or epinephrine signal, the G_M protein can be phosphorylated at two different sites. 1) Phosphorylation of G_M at site 1, stimulated by insulin, activates PP1, which dephosphorylates phosphorylase kinase, glycogen phosphorylase, and glycogen synthase. 2) Phosphorylation of G_M at site 2, triggered by epinephrine, causes the dissociation of PP1 from the glycogen particle, blocking its access to glycogen phosphorylase and glycogen synthase. PKA also phosphorylates inhibitor 1, which in its phosphorylated state inhibits PP1. Thus, insulin inhibits glycogen breakdown and stimulates its synthesis, whereas epinephrine (or glucagon in the liver) exerts the opposite effect.

Allosteric and hormonal signals coordinate Carbohydrate Metabolism

Having examined the mechanisms governing the operation of individual enzymes, we can now turn to the alterations in carbohydrate metabolism that occur during the fed state, fasting, and the fight-or-flight response, signaled by insulin, glucagon, and epinephrine, respectively. We will consider two scenarios in which regulation is directed toward different goals: 1) supplying glucose to the blood via hepatocytes; 2) utilizing CARBOHYDRATES as an energy source by other tissues (specifically, Skeletal Muscle myocytes) to meet their own energy demands.

Following a carbohydrate-rich meal, blood glucose levels rise, leading to the release of insulin (upper part of Fig. 15-41). In hepatocytes, insulin immediately exerts two effects: it inactivates GSK3 via the cascade depicted in Fig. 15-39, and it activates a protein phosphatase, likely PP1. These two actions fully activate glycogen synthase. In addition, through dephosphorylation by PP1, insulin inactivates glycogen phosphorylase a and phosphorylase kinase, thereby halting glycogen breakdown. Glucose enters hepatocytes via GLUT2, which is constitutively present in the plasma membrane; elevated intracellular glucose levels trigger the dissociation of hexokinase IV (glucokinase) from its complex with a nuclear regulatory protein. Hexokinase IV translocates to the cytosol and phosphorylates glucose, stimulating glycolysis and providing substrate for glycogen synthesis. Under these conditions, hepatocytes utilize blood glucose to synthesize glycogen, The amount of which can reach up to 10% of the total liver mass.

Between meals or during prolonged fasting, a drop in blood glucose levels triggers the release of glucagon, which activates PKA through the cascade mechanism shown in Fig. 15-35. PKA mediates all the effects exerted by glucagon (lower region of Fig. 15-41): it phosphorylates phosphorylase kinase, activating it and subsequently leading to the activation of glycogen phosphorylase; it phosphorylates glycogen synthase, inactivating it and halting glycogen synthesis; it phosphorylates the bifunctional enzyme PFK-2/FBPase-2, resulting in a decreased concentration of fructose-2,6-bisphosphate, which in turn leads to the inactivation of the glycolytic enzyme PFK-1 and the activation of the gluconeogenic enzyme FBPase-1; finally, PKA phosphorylates and inactivates the glycolytic enzyme Pyruvate kinase. Under these conditions, glycogen breakdown and Gluconeogenesis in the liver generate glucose-6-phosphate, The Use of glucose for energy via glycolysis or for glycogen synthesis is halted, and all these processes combine to increase the net output of glucose from the liver into the bloodstream.

Fig. 15-41. REGULATION OF CARBOHYDRATE Metabolism in hepatocytes. Arrows indicate causal relationships between occurring changes. The notation ↓A -> ↑B means that a decrease in A causes an increase in B. Pink arrows denote events occurring during high blood glucose levels; blue arrows denote events during low blood glucose.

Such glucose release is possible only in the liver because other tissues lack glucose-6-phosphatase (Fig. 15-28).

In terms of Metabolic Regulation, skeletal muscle physiology differs from liver physiology in three important respects (Fig. 15-42). 1) Muscles use their stored glycogen exclusively for their own energy needs. 2) The transition from rest to active muscle contraction drastically alters the ATP demands of muscles, which are met by glycolysis. 3) Muscles lack the necessary enzymatic machinery to carry out gluconeogenesis. The regulation of Carbohydrate Metabolism in muscles directly reflects these differences from liver metabolism. First, myocytes lack the glucagon receptor. Second, the muscle isoform of pyruvate kinase is not phosphorylated by PKA, so glycolysis is not shut down by high cAMP concentrations. In fact, cAMP accelerates The rate of glycolysis in muscles, likely by activating glycogen phosphorylase. When epinephrine is released into the bloodstream during the fight-or-flight response, the elevated cAMP concentration leads to the activation of PKA, which phosphorylates and activates glycogen phosphorylase kinase. The phosphorylation and activation of glycogen phosphorylase result in an accelerated rate of glycogen breakdown. During mild stress, epinephrine is not released, but upon any neuronal stimulation of muscles, the cytosolic Ca²⁺ concentration increases rapidly, which activates phosphorylase kinase via its calmodulin subunit.

Fig. 15-42. Differential regulation of carbohydrate metabolism in the liver and muscles. In the liver, either glucagon (during low blood glucose) or epinephrine (in a fight-or-flight situation) triggers a maximal release of glucose into the bloodstream. In muscles, epinephrine enhances glycogen breakdown and glycolysis, and these processes provide the ATP required for muscle contraction.

An elevated insulin level leads to enhanced glycogen synthesis in myocytes through the activation of PP1 and the inactivation of GSK3. Unlike hepatocytes, myocytes sequester GLUT4 in intracellular vesicles. Insulin stimulates the translocation of these vesicles to the plasma membrane, where they enhance glucose uptake. Consequently, in response to an insulin signal, myocytes help lower blood glucose levels by increasing glucose uptake, glycogen synthesis, and glycolysis.

Carbohydrate and Lipid Metabolism are linked by hormonal and allosteric regulatory mechanisms

Complex though the regulation of carbohydrate metabolism is, it represents only one component of overall Energy Metabolism. The metabolism of fats and Fatty acids is intimately intertwined with carbohydrate metabolism. Hormonal signals such as insulin, dietary changes, and Physical Exercise exert equally profound effects on the Regulation of Lipid Metabolism and its coordination with carbohydrate metabolism. We will explore mammalian systemic metabolism in Chapter 23, after first reviewing the pathways of lipid and Amino acid metabolism (Chapters 17 and 18). Here, we merely wish to emphasize that Metabolic pathways are subject to strict control that is remarkably sensitive to shifts in the metabolic state. Regulatory mechanisms are designed to match the flux of metabolites through metabolic pathways to the immediate needs of The Cell and the Organism while minimizing fluctuations in the concentrations of intermediates operating in other pathways.

Summary of Section 15.5 Coordinated Regulation of Glycogen Synthesis and Breakdown

■ Glycogen Phosphorylase is activated in response to glucagon or adrenaline, which increases the concentration of cGMP and activates PKA. Subsequently, PKA phosphorylates and activates phosphorylase kinase, which converts glycogen phosphorylase b into the active form a. Phosphoprotein phosphatase 1 (PP1) catalyzes the reverse reaction, inactivating phosphorylase a. Glucose binds to the hepatic isoenzyme of glycogen phosphorylase a, promoting its dephosphorylation and inactivation.

■ Glycogen synthase a is inactivated by phosphorylation catalyzed by GSK3. Insulin blocks the action of GSK3. Insulin-activated PP1 relieves this inhibition by dephosphorylating glycogen synthase b.

■ Insulin enhances glucose uptake by myocytes and adipocytes by promoting the translocation of the GLUT4 glucose transporter to the plasma membrane.

■ Insulin stimulates the synthesis of hexokinases II and IV, PFK-1, pyruvate kinase, and several enzymes involved in lipid synthesis. Insulin also stimulates glycogen synthesis in the liver and muscles.

■ In the liver, glucagon stimulates glycogen breakdown and gluconeogenesis while inhibiting glycolysis, thereby conserving glucose for the needs of the Brain and other tissues.

■ In muscles, adrenaline stimulates glycogen breakdown and glycolysis, supplying cells with ATP for muscle contraction.