Voet D., Voet Ju.G. Biochemistry

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b. Second Messengers Mediate Glucagon- and

Epinephrine-Stimulated Glycogen Breakdown

The response to glucagon and epinephrine involves the

release inside the cell of molecules known as second messen-

gers, that is, intracellular mediators of the externally received

hormonal message. Different receptors act to release dif-

ferent second messengers. Indeed, cAMP was identified by

Earl Sutherland as the first known instance of a second

messenger through his demonstration that glucagon and

epinephrine act at cell surfaces to stimulate adenylyl cy-

clase (AC) to increase [cAMP] [the mechanism of AC acti-

vation, as well as a discussion of other second messengers,

including Ca

2⫹

, inositol-1,4,5-trisphosphate (IP

), and

diacylglycerol (DAG), is elaborated in Sections 19-2D and

19-4A]. Following this discovery, it was realized that

cAMP, which is present in all forms of life, is an essential

control element in many biological processes.

When hormonal stimulation by glucagon or epineph-

rine increases the intracellular cAMP concentration, the

protein kinase A activity increases, increasing the rates of

phosphorylation of many proteins and decreasing their

dephosphorylation rates as well. A decrease in dephos-

phorylation rates, as previously noted, increases the phos-

phorylation level of phosphoprotein phosphatase in-

hibitor-1, which in turn inhibits phosphoprotein

phosphatase-1. An increase in the concentration of phos-

phorylase a also contributes to the inhibition of phospho-

protein phosphatase-1.

Because of the amplifying properties of the cyclic cas-

cades, a small change in [cAMP] results in a large change in

the fraction of enzymes in their phosphorylated forms.

When a large fraction of the glycogen metabolism enzymes

are present in their phosphorylated forms, the metabolic

flux is in the direction of glycogen breakdown, since glyco-

gen phosphorylase is active and glycogen synthase is inac-

tive. When [cAMP] decreases, phosphorylation rates de-

crease, dephosphorylation rates increase, and the fraction

of enzymes in their dephospho forms increases. The result-

ant activation of glycogen synthase and the inhibition of

glycogen phosphorylase cause a change in the flux direc-

tion toward net glycogen synthesis.

F. Maintenance of Blood Glucose Levels

An important function of the liver is to maintain the blood

concentration of glucose, the brain’s primary fuel source, at

⬃5 mM.When blood [glucose] decreases beneath this level,

usually during exercise or well after meals have been di-

gested, the liver releases glucose into the bloodstream. The

process is mediated by the hormone glucagon as follows:

1. Glucose inhibits the pancreatic ␣ cells from secreting

glucagon into the bloodstream. When the blood glucose

concentration falls, this inhibition is released causing the ␣

cells to secrete glucagon.

2. Glucagon receptors on liver cell surfaces respond to

the presence of glucagon by activating adenylate cyclase,

thereby increasing the [cAMP] inside these cells.

3. The [cAMP] increase, as described above, triggers an

increase in the rate of glycogen breakdown, leading to in-

creased intracellular [G6P].

4. G6P, in contrast to glucose, cannot pass through the

cell membrane. However, in liver, which does not employ

glucose as a major energy source, the enzyme glucose-6-

phosphatase (G6Pase) hydrolyzes G6P:

The resulting glucose enters the bloodstream, thereby in-

creasing the blood glucose concentration. Muscle and brain

cells, however, lack G6Pase so that they retain their G6P.

G6P hydrolysis requires intracellular G6P transport.

G6P is produced in the cytosol, whereas G6Pase is a TM

protein that resides in the endoplasmic reticulum (ER)

membrane with its active site in the ER lumen. G6P must

therefore be imported into the ER by a G6P translocase

before it can be hydrolyzed. The resulting glucose and P

are then returned to the cytosol via specific transport pro-

teins (Section 18-3G).A defect in any of the components of

this G6P hydrolysis system results in type I glycogen stor-

age disease (Section 18-4).

How does this delicately balanced system respond to

an increase in blood [glucose]? When blood sugar is high,

normally immediately after meals have been digested,

glucagon levels decrease and insulin is released from

the pancreatic ␤ cells. The rate of glucose transport

across many cell membranes increases in response to in-

sulin (through the insulin-dependent glucose transporter

GLUT4; Section 20-2E), [cAMP] decreases, and glycogen

metabolism therefore shifts from glycogen breakdown to

glycogen synthesis. The mechanism of insulin action is

quite complex and is not fully understood (Sections 19-3

and 19-4F), but one of its target enzymes appears to be

phosphoprotein phosphatase-1.

In muscle, insulin and epinephrine have antagonistic ef-

fects on glycogen metabolism. Epinephrine promotes

glycogenolysis by activating the cAMP-dependent phos-

phorylation cascade, which stimulates glycogen breakdown

while inhibiting glycogen synthesis. Insulin, as we saw in

Section 18-3Cg, activates insulin-stimulated protein kinase

to phosphorylate site 1 on the glycogen-binding G

sub-

unit of phosphoprotein phosphatase-1 so as to activate this

protein to dephosphorylate the enzymes of glycogen me-

tabolism (Fig. 18-22). The storage of glucose as glycogen is

thereby stimulated through the inhibition of glycogen

breakdown and the stimulation of glycogen synthesis.

In liver,it appears that glucose and glucose-6-phosphate

themselves may be the messengers to which the glycogen

metabolism system responds. Glucose inhibits phosphory-

lase a by binding only to the active site of the enzyme’s inac-

tive T state, but in a manner different from that of substrate.

The presence of glucose therefore shifts phosphorylase a’s

T 34 R equilibrium toward the T state (Fig. 18-10, right).

This conformational shift, as we saw in Section 18-3Cg, ex-

poses the Ser 14-phosphoryl group to phosphoprotein

phosphatase-1, resulting in the demodification of phospho-

rylase a. An increase in glucose concentration therefore

G6P ⫹ H

glucose ⫹ P

Section 18-3. Control of Glycogen Metabolism 661

JWCL281_c18_638-670.qxd 6/3/10 1:49 PM Page 661

promotes inactivation of glycogen phosphorylase a

through the enzyme’s conversion to phosphorylase b

(Fig. 18-23; i.e., phosphorylase a acts as a glucose receptor).

The concomitant release of phosphoprotein phosphatase-1

inhibition (recall that it specifically binds to and is thereby

inactivated by phosphorylase a), moreover, results in the

activation (dephosphorylation) of m-glycogen synthase b.

In addition,glucose is converted to G6P by glucokinase (see

below), which facilitates the dephosphorylation and activa-

tion of m-glycogen synthase b to o-glycogen synthase a.

Above a glucose concentration of 7 mM, these processes

reverse the flux of glycogen metabolism from breakdown to

synthesis. The liver can thereby store the excess glucose as

glycogen.

a. Glucokinase Forms G6P at a Rate Proportional

to the Glucose Concentration

The liver’s function in “buffering” the blood [glucose] is

made possible because this organ contains a variant of hex-

okinase (the first glycolytic enzyme; Section 17-2A) known

as glucokinase (GK; also called hexokinase D and

hexokinase IV). The hexokinase in most cells obeys

Michaelis–Menten kinetics, has a high glucose affinity

 0.1 mM; the value of [glucose] at which the enzyme

achieves half-maximal velocity; Section 14-2A), and is in-

hibited by its reaction product, G6P. GK, in contrast, has a

much lower glucose affinity (reaching half of its maximal

velocity at ⬃5 mM) and displays sigmoidal kinetics with a

Hill constant (Section 10-1Bb) of 1.5, so its activity in-

creases rapidly with the blood [glucose] over the normal

physiological range (Fig. 18-24; see Problem 7 of this chap-

ter). GK, moreover, is not inhibited by physiological concen-

trations of G6P. Consequently, the higher the blood [glu-

cose], the faster the liver converts glucose to G6P [liver

cells, unlike most cells, contain ample quantities of the

insulin-independent glucose transporter GLUT2 (Section

20-2Eb) and are therefore freely permeable to glucose;

their glucose transport rate is unresponsive to insulin].

Thus at low blood [glucose], the liver does not compete

with other tissues for the available glucose supply, whereas

at high blood [glucose],when the glucose needs of these tis-

sues are met, glucose in the liver is converted to G6P. The

excess glucose in the liver induces the inactivation of glyco-

gen phosphorylase and the release of phosphoprotein

phosphatase-1, whereas the resulting G6P allosterically

facilitates the activation of glycogen synthase via dephos-

phorylation. The net result is that the liver converts the

excess glucose to glycogen. (Note that GK is a monomeric

enzyme, so that its sigmoidal rate increase with [glucose]

is a puzzling observation in light of the various allosteric

models indicating that monomeric enzymes are inca-

pable of cooperative behavior. Since GK does not exhibit

Michaelis–Menten kinetics, the glucose concentration

when this enzyme has half its maximal activity is known as

its K

0.5

in analogy with the operational definition of K

GK is subject to metabolic controls. Emile Van Schaftin-

gen isolated glucokinase regulatory protein (GKRP; a

625-residue monomer) from rat liver, which, in the pres-

ence of the glycolytic intermediate fructose-6-phosphate

(F6P), is a competitive inhibitor of glucokinase. Fructose-1-

phosphate (F1P), an intermediate in liver fructose metabo-

lism (Section 17-5A), overcomes this inhibition. Since

fructose is normally available only from dietary sources

(e.g., sucrose), fructose may be the signal that triggers the

uptake of dietary glucose by the liver.

662 Chapter 18. Glycogen Metabolism

Figure 18-23 The enzymatic activities of phosphorylase a and

glycogen synthase in mouse liver in response to an infusion of

glucose. Phosphorylase a is rapidly inactivated and, somewhat

later, glycogen synthase is activated. [After Stalmans, W., De

Wulf, H., Hue, L., and Hers, H.-G., Eur. J. Biochem. 41, 129

(1974).]

Figure 18-24 Comparison of the relative enzymatic activities

of hexokinase and glucokinase over the physiological blood

glucose range. The affinity of glucokinase for glucose

0.5

 5 mM ) is much lower than that of hexokinase

 0.1 mM) and exhibits sigmoidal rather than hyperbolic

variation with [glucose]. [The glucokinase curve was generated

using the Hill equation (Eq. [10.7]) with K  10 mM and

n  1.5 as determined by Cardenas, M.L., Rabajille, E., and

Niemeyer, H., Eur. J. Biochem. 145, 163–171 (1984).]

04826

Phosphorylase a

Glycogen

synthase

Time after glucose infusion (min)

Enzyme activity

Hexokinase

Glucokinase

0.0

0.2

0.4

0.6

0.8

1.0

[Glucose] (mM)

max

10 15

JWCL281_c18_638-670.qxd 2/26/10 2:24 PM Page 662

b. Glucokinase Regulates Glucose Homeostasis via

an Intracellular Localization Mechanism

Intracellular localization plays an important role in GK

inhibition by GKRP. GK translocates freely between the

nucleus and cytoplasm. However, GKRP is located exclu-

sively in the nucleus. When the glucose concentration is

low, GK remains bound to GKRP in the nucleus, where it is

unavailable to phosphorylate glucose. At increased con-

centrations of glucose and/or F1P, GK dissociates from

GKRP and translocates to the cytoplasm, where it phos-

phorylates glucose to G6P, starting it on its way toward

glycogen synthesis.The antagonism of GK and GKRP pro-

vides a major mechanism for controlling glucose phospho-

rylation and glycogen metabolism in the liver. Their flux

control coefficients (Section 17-4Ca) in liver cells are close

to ⫹1 for GK and ⫺1 for GKRP (a negative flux control

coefficient is indicative of inhibition). This countervailing

relationship provides a sensitive mechanism for maintain-

ing glucose homeostasis.

Phosphoglucomutase, which has a high enough activity

to equilibrate its substrate and product and therefore func-

tions in either direction, transforms G6P to G1P, which is

then converted to glycogen. Some of the G6P is also recon-

verted to glucose by the action of glucose-6-phosphatase in

what amounts to a “futile” cycle. This is apparently the en-

ergetic price of effective glucose “buffering” of the blood.

c. Fructose-2,6-Bisphosphate Activates Glycolysis

␤-D-Fructose-2,6-bisphosphate (F2,6P)

is also an important factor in the liver’s maintenance of

blood [glucose]. F2,6P, which is not a glycolytic metabolite,

is an extremely potent allosteric activator of animal phos-

phofructokinase (PFK) and an inhibitor of fructose bispho-

sphatase (FBPase). F2,6P, which was independently discov-

HHO

HO H

–2

2–

␤-D-Fructose-2,6-bisphosphate

(F2,6P)

ered in 1980 by Simon Pilkis, by Emile Van Schaftingen and

Henri-Géry Hers, and by Kosaku Uyeda, therefore stimu-

lates glycolytic flux (the F6P–FBP substrate cycle is dis-

cussed in Section 17-4Ff).

The concentration of F2,6P in the cell depends on the bal-

ance between its rates of synthesis and degradation by phos-

phofructokinase-2 (PFK-2; also called 6PF-2-K) and fruc-

tose bisphosphatase-2 (FBPase-2; also called F-2,6-Pase),

respectively (Fig. 18-25). These enzyme activities are located

on different domains of a single ⬃100-kD homodimeric

protein named PFK-2/FBPase-2. The X-ray structure of the

H256A mutant of rat testis PFK-2/FBPase-2 in complex

with F6P, P

, succinate, and the nonhydrolyzable ATP ana-

log adenosine-5ⴕ-(␤,␥-imido)triphosphate (AMPPNP)

was determined by Uyeda and Charles Hasemann (Fig. 18-

26). It indicates, in agreement with a variety of studies,

that the PFK-2 activity resides on each subunit’s 246-residue

N-terminal domain, whereas the FBPase-2 activity resides

on each subunit’s 213-residue C-terminal domain. The

succinate, which binds in the vicinity of AMPPNP’s ␥ phos-

phate group, presumably occupies the F6P binding pocket in

the PFK-2 active site, whereas the F6P and P

mark the F2,6P

binding site in the FBPase-2 active site. The FBPase-2 do-

main is structurally related to the glycolytic enzyme phos-

phoglycerate mutase (PGM; Section 17-2H) and shares a

common catalytic mechanism involving a covalent phospho-

His intermediate (His 256 in FBPase-2).The PFK-2 domain

is structurally related to adenylate kinase (Section 17-4Fe)

but not, as had been speculated, to PFK (also called PFK-1

to distinguish this glycolytic enzyme from PFK-2).

The enzyme activities of PFK-2/FBPase-2 are subject to

allosteric regulation by a variety of metabolic intermediates

–

Adenosine-5ⴕ-(␤,␥-imido)triphosphate

(AMPPNP)

OH OH

–

P NH

Section 18-3. Control of Glycogen Metabolism 663

Figure 18-25 Formation and degradation of ␤-D-fructose-2,

6-bisphosphate as catalyzed by PFK-2 and FBPase-2. These two

enzyme activities occur on different domains of the same protein

HHO

ATP ADP

liver PFK-2

(dephosphoenzyme)

liver FBPase-2

(phosphoenzyme)

β-

D-Fructose-6-phosphate

(F6P)

–

HHO

β-D-Fructose-2,6-bisphosphate

(F2,6P)

–

molecule. Dephosphorylation of the liver enzyme activates

PFK-2 but deactivates FBPase-2.

JWCL281_c18_638-670.qxd 6/30/10 12:00 PM Page 663

as well as to phosphorylation/dephosphorylation by pro-

tein kinase A and a phosphoprotein phosphatase. Phos-

phorylation of the liver enzyme at its Ser 32 inhibits its

PFK-2 activity and activates its FBPase-2 activity.Thus, the

pancreatic  cell’s release of glucagon in response to low

blood [glucose] results, through an increase in liver

[cAMP], in a decreased liver [F2,6P]. This situation, in turn,

decreases the PFK-1 activity, thereby inhibiting glycolysis.

Hence, the G6P resulting from the concurrent stimulation

of glycogen degradation is converted to glucose and se-

creted as described above rather than being metabolized.

Simultaneously, the deinhibition of FBPase (also called

FBPase-1 to distinguish it from FBPase-2) by the decrease

of [F2,6P] stimulates gluconeogenesis, the formation of

glucose from nonglucose precursors such as amino acids by

a pathway that effectively reverses glycolytic flux (and in

which FBPase-1 is a key regulatory enzyme; Section 23-1).

This process provides a second means of glucose produc-

tion. Conversely, when the blood [glucose] is high, cAMP

levels decrease, the liver PFK-2/FBPase-2 is dephosphory-

lated by phosphoprotein phosphatase-1 activating PFK-2,

which, in turn, causes a rise in [F2,6P]. PFK-1 is therefore

activated, FBPase-1 is inhibited, and the net glycolytic flux

changes from gluconeogenesis to glycolysis.

The F2,6P control systems in skeletal muscle and in

heart muscle function quite differently from that in liver due

to the presence in these tissues of different PFK-2/FBPase-2

isozymes. In heart and skeletal muscles, increased glyco-

gen breakdown is coordinated with increased glycolysis

rather than increased glucose secretion. This is because

phosphorylation of the heart muscle PFK-2/FBPase-2

isozyme occurs at entirely different sites (Ser 406 and Thr

475 of the 530-residue protein) from that of the liver

isozyme (Ser 32 of the 470-residue protein) and activates

rather than inhibits PFK-2. Consequently, hormones that

stimulate glycogen breakdown also increase heart muscle

[F2,6BP], thereby stimulating glycolysis as well. The skele-

tal muscle and testis isozymes lack phosphorylation

sites altogether and are therefore not subject to cAMP-

dependent phosphorylation control.

G. Response to Stress

Epinephrine and norepinephrine, which are often called

the “fight or flight” hormones, are released into the blood-

stream by the adrenal glands in response to stress. Epi-

nephrine receptors (known as ␤-adrenergic receptors; Sec-

tion 19-1F) present on the surfaces of liver and muscle cells

respond to these hormones just as glucagon receptors re-

spond to the presence of glucagon; they activate adenylate

cyclase, thereby increasing intracellular [cAMP]. Indeed,

epinephrine also stimulates the pancreatic  cells to re-

lease glucagon, which further increases liver [cAMP]. The

G6P produced by the consequent glycogen breakdown in

muscle enters the glycolytic pathway, thereby generating

ATP and helping the muscles cope with the stress that trig-

gered the epinephrine release.

The liver’s response to stress, in addition to its response

to the glucagon released by pancreatic epinephrine stimu-

lation, involves response to epinephrine stimulation via

two types of receptors, -adrenergic receptors, as discussed

above, and ␣-adrenergic receptors. -Adrenergic receptors

act by stimulating phospholipase C to release other second

messengers, namely inositol-1,4,5-trisphosphate (IP

), dia-

cylglycerol (DAG), and Ca

2

(Fig. 18-27a), which act to re-

inforce the cells’ response to cAMP. As we mentioned in

Section 18-3Cf, phosphorylase kinase, which both activates

phosphorylase and inactivates glycogen synthase, is fully

active only when both phosphorylated and in the presence

of increased [Ca

2

]. In addition, glycogen synthase is inac-

tivated by phosphorylation by several other Ca

2

-depend-

ent protein kinases, including protein kinase C (Section

18-3D). Protein kinase C requires both Ca

2

and DAG for

activity (Section 19-4C).This dual stimulation of receptors

in response to epinephrine causes the liver to produce

664 Chapter 18. Glycogen Metabolism

Figure 18-26 X-ray structure of the H256A mutant of rat

testis PFK-2/FBPase-2. The N-terminal PFK-2 domain is blue

and the C-terminal FBPase-2 domain is yellow-green.The bound

2

–AMPPNP, succinate, F6P, and P

are shown in space-filling

form colored according to atom type (AMPPNP C green,

succinate C magenta, F6P C cyan, N blue, O red, Mg

2

light

green, and P orange). The P

, which occupies the binding site of

the F2,6P’s 2-phosphate group, is opposite the site that would be

occupied by the side chain of the wild-type enzyme’s His 256

(magenta), to which it would be transferred in the catalytic

reaction.The succinate occupies the presumed F6P-binding

pocket of the PFK-2 domain. [Based on an X-ray structure by

Kosaku Uyeda and Charles Hasemann, University of Texas

Southwestern Medical Center. PDBid 2BIF.]

JWCL281_c18_638-670.qxd 2/26/10 2:24 PM Page 664

Section 18-3. Control of Glycogen Metabolism 665

Phosphatidylinositol-4,5-bisphosphate (PIP

)

–

OPO

2–

OPO

2–

1,2-Diacylglycerol

(DAG)

Inositol-1,4,5-triphosphate

(IP

)

–

OPO

2–

OPO

2–

⫹

phospholipase C

(a)

β-Adrenergic

receptor

Glucagon

receptor

Epinephrine Glucagon

glycogen

synthase

phospho-

lipase C

adenylate

cyclase

adenylate

cyclase

ATP

Liver

Cell

ATP

PIP

DAG

UDP–glucose

pyrophosphorylase

glycogen

phosphorylase

cAMP

Glycogen

G1P

G6P

UDP–glucose

phosphoglucomutase

Glucose-6-

phosphatase

GLUT2 glucose

transporter

α-Adrenergic receptor

Glucose

G6P

Glucose

Endoplasmic

reticulum

(b)

Figure 18-27 The liver’s response to

stress. (a) Stimulation of ␣-adrenergic

receptors by epinephrine activates

phospholipase C to hydrolyze

phosphatidylinositol-4,5-bisphosphate

(PIP

) to inositol-1,4,5-trisphosphate

(IP

) and diacylglycerol (DAG). (b) The

participation of two second messenger

systems: the cAMP-mediated stimulation

of glycogenolysis and inhibition of

glycogen synthesis triggered by glucagon

and ␤-adrenergic receptor activation; and

the IP

, DAG, and Ca

2⫹

-mediated

stimulation of glycogenolysis and

inhibition of glycogen synthesis triggered

by ␣-adrenergic receptor activation. IP

stimulates the release of Ca

2⫹

from the

endoplasmic reticulum, whereas DAG,

together with Ca

2⫹

, activates protein

kinase C to phosphorylate and thereby

inactivate glycogen synthase. G6Pase

occupies the endoplasmic reticulum.

Consequently, the cytosolically produced

G6P is transported into the endoplasmic

reticulum via the T1 G6P translocase,

where it is hydrolyzed to glucose and P

The P

and glucose are then returned to

the cytosol by the T2 and T3 transporters,

respectively, and the glucose is exported

from the cell via the GLUT2 glucose

transporter.

JWCL281_c18_638-670.qxd 6/10/10 1:09 PM Page 665

G6P, which is hydrolyzed by G6Pase, resulting in the re-

lease of glucose into the bloodstream, thereby further fuel-

ing the muscles (Fig. 18-27b).

4 GLYCOGEN STORAGE DISEASES

With glycogen metabolism being such a finely controlled

system, it is not surprising that genetically determined en-

zyme deficiencies result in disease states. The study of

these disease states and the enzyme deficiencies that cause

them has provided insights into the system’s balance. In

this sense, genetic diseases are valuable research tools.

Conversely, the biochemical characterization of the path-

ways affected by a genetic disease often leads, as we shall

see, to useful strategies for its treatment. Many diseases

have been characterized that result from inherited defi-

ciencies of one or another of the enzymes of glycogen me-

tabolism. These defects are listed in Table 18-1 and dis-

cussed in this section.

Type I: Glucose-6-Phosphatase Deficiency

(von Gierke’s Disease)

G6Pase catalyzes the final step leading to the release of

glucose into the bloodstream by the liver. Deficiency of

this enzyme results in an increase of intracellular [G6P],

which leads to a large accumulation of glycogen of normal

structure in the liver and kidney (recall that G6P inhibits

glycogen phosphorylase and activates glycogen synthase)

and an inability to increase blood glucose concentration in

response to glucagon or epinephrine. Similar difficulties

occur when there are defects in the protein that transports

glucose across the liver cell plasma membrane (Section

20-2Eb) or in any of the proteins that transport glucose,

G6P, or P

across the endoplasmic reticulum membrane

(Section 18-3F; Fig. 18-27b). The symptoms of Type I

glycogen storage disease include massive liver enlarge-

ment, severe hypoglycemia (low blood sugar) after a few

hours’ fast, and a general failure to thrive. Treatment of

the disease has included drug-induced inhibition of glu-

cose uptake by the liver to increase blood [glucose], con-

tinuous intragastric feeding overnight, again to increase

blood [glucose], oral administration of uncooked corn

starch (which is only slowly broken down to glucose), and

surgical transposition of the portal vein, which ordinarily

feeds the liver directly from the intestines, so as to allow

this glucose-rich blood to reach peripheral tissues before it

reaches the liver.This latter treatment has the added ben-

efit of allowing the tissues to receive more glucose while

decreasing the storage of this glucose as liver glycogen.

Liver transplantation has also been successful in the few

patients in which this treatment has been tried.

A gene therapy protocol (Section 5-5H) is being devel-

oped to correct type I glycogen storage disease. G6Pase-

deficient knockout mice (Section 5-5H) have been treated

with a viral vector containing the mouse G6Pase gene.This

treatment, which delivers G6Pase to the livers of these

mice, greatly increases their survival rate and corrects the

metabolic abnormalities associated with this glycogen stor-

age disease.

Type II: ␣-1,4-Glucosidase Deficiency

(Pompe’s Disease)

This is the most devastating glycogen storage disease. It

results in a large accumulation of glycogen of normal

structure in the lysosomes of all cells and causes death by

cardiorespiratory failure, usually before the age of 1 year.

We have not discussed ␣-1,4-glucosidase in the sections on

the pathways of glycogen synthesis and breakdown since it

is not among those enzymes. It occurs in lysosomes, where

it functions to hydrolyze the disaccharide maltose (Section

11-2B) and linear oligosaccharides, as well as the outer

branches of glycogen, thereby yielding free glucose. How-

ever, this second pathway of glycogen metabolism is not

quantitatively important. The reason that lysosomes nor-

mally take up and degrade glycogen granules is unknown.

666 Chapter 18. Glycogen Metabolism

Table 18-1 Hereditary Glycogen Storage Diseases

Type Enzyme Deficiency Tissue Common Name Glycogen Structure

I Glucose-6-phosphatase Liver von Gierke’s disease Normal

II ␣-1,4-Glucosidase All lysosomes Pompe’s disease Normal

III Amylo-1,6-glucosidase All organs Cori’s disease Outer chains missing

(glycogen debranching enzyme) or very short

IV Amylo-(1,4 S 1,6)- Liver, probably Andersen’s disease Very long

transglycosylase all organs unbranched chains

(glycogen branching enzyme)

V Glycogen phosphorylase Muscle McArdle’s disease Normal

VI Glycogen phosphorylase Liver Hers’ disease Normal

VII Phosphofructokinase Muscle Tarui’s disease Normal

VIII Phosphorylase kinase Liver X-Linked phosphorylase Normal

kinase deficiency

IX Phosphorylase kinase All tissues Normal

0 Glycogen synthase Liver Normal, deficient

in quantity

JWCL281_c18_638-670.qxd 6/30/10 12:00 PM Page 666

Type III: Amylo-1,6-Glucosidase (Glycogen

Debranching Enzyme) Deficiency (Cori’s Disease)

In this disease, glycogen of abnormal structure contain-

ing very short outer chains accumulates in both liver and

muscle since, in the absence of debranching enzyme, the

glycogen cannot be further degraded. Its hypoglycemic

symptoms are similar to, but not as severe as, those of von

Gierke’s disease (Type I). The low blood sugar, which in

this case is a result of the decreased efficiency of glycogen

breakdown, is treated with frequent feeding and a high-

protein diet [in response to low blood sugar, the liver,

through gluconeogenesis (Section 23-1), synthesizes glucose

from amino acids]. For unknown reasons, the symptoms of

Cori’s disease often disappear at puberty.

Type IV: Amylo-(1,4 S 1,6)-Transglycosylase

(Glycogen Branching Enzyme) Deficiency

(Andersen’s Disease)

This is one of the most severe glycogen storage diseases;

victims rarely survive past the age of 5 years because of

liver dysfunction. Glycogen concentration in liver is not in-

creased but its structure is abnormal, with very long un-

branched chains resulting from the lack of glycogen

branching enzyme. This decreased branching greatly

reduces the solubility of glycogen. It has been suggested

that the liver dysfunction may be caused by a “foreign

body” immune reaction to the abnormal glycogen.

Type V: Muscle Glycogen Phosphorylase Deficiency

(McArdle’s Disease)

We have mentioned this condition in connection with

the realization that glycogen synthesis and breakdown

must occur by different pathways (Section 18-2). Its major

symptom, which is most severely manifested in early adult-

hood, is painful muscle cramps on exertion.This situation is

a result of the inability of the glycogen breakdown system

to provide sufficient fuel for glycolysis to keep up with the

metabolic demand for ATP. Studies by

P NMR on human

forearm muscle have noninvasively corroborated this con-

clusion by demonstrating that exercise in individuals with

McArdle’s disease leads to elevated muscle ADP levels

compared to those of normal individuals (Fig. 18-28). Curi-

ously, if McArdle’s victims continue their exertions after a

short rest, their cramps subside. This “second wind” effect

has been attributed to the muscles’ switch from anaerobic

to aerobic metabolism as well as vasodilation, which gives

the muscles increased access to the glucose and fatty acids

in the blood for use as alternative fuels to glycogen. Liver

glycogen phosphorylase is normal in these individuals, im-

plying the presence of different glycogen phosphorylase

isozymes in muscle and liver.

Type VI: Liver Glycogen Phosphorylase Deficiency

(Hers’ Disease)

Patients with a deficiency of liver phosphorylase have

symptoms similar to those with mild forms of Type I glyco-

gen storage disease. The hypoglycemia in this case results

from the inability of glycogen phosphorylase to respond to

the need for glucose production by the liver.

Type VII: Muscle Phosphofructokinase Deficiency

(Tarui’s Disease)

The result of a deficiency of the glycolytic enzyme PFK-

1 in muscle is an abnormal buildup of the glycolytic

metabolites G6P and F6P. High concentrations of G6P in-

crease the activities of glycogen synthase (G6P activates

glycogen synthase and inactivates glycogen phosphory-

lase) and UDP–glucose pyrophosphorylase (G6P is in

equilibrium with G1P, a substrate for the enzyme) so that

glycogen accumulates in muscle. Other symptoms are

similar to those of Type V glycogen storage disease, mus-

cle phosphorylase deficiency, since PFK deficiency pre-

vents glycolysis from keeping up with the ATP demand of

muscle contraction.

Type VIII: Liver Phosphorylase Kinase Deficiency

(X-Linked Phosphorylase Kinase Deficiency)

Some individuals with symptoms of Type VI glycogen

storage disease have liver phosphorylase of normal struc-

ture. However, they have a defective phosphorylase kinase,

which results in their inability to convert phosphorylase b

to phosphorylase a. The gene encoding the  subunit of

phosphorylase kinase resides on the X chromosome and

hence Type VIII disease is X-linked rather than autosomal,

as are the other glycogen storage diseases.

Type IX: Phosphorylase Kinase Deficiency

Phosphorylase kinase deficiency, when it is autosomal

recessive, is caused by a mutation in one of the genes

encoding a , , or  subunit of phosphorylase kinase. Since

different organs contain different isozymes of phosphory-

lase kinase, the symptoms and severity of Type IX disease

vary with the affected organs.

Section 18-4. Glycogen Storage Diseases 667

Figure 18-28 The ADP concentration in human forearm

muscles during rest and following exertion in normal individuals

and those with McArdle’s disease. The ADP concentration was

determined from

P NMR measurements on intact forearms.

[After Radda, G.K., Biochem. Soc.Trans. 14, 522 (1986).]

McArdle's

patients

Normal

individuals

Rest

Light exercise

Rest

Upon cramps after light exercise

After “second wind”

Heavy exercise

leading to exhaustion

080

[ADP] (μM)

16040 120 200180

JWCL281_c18_638-670.qxd 2/26/10 2:24 PM Page 667

1 Glycogen Breakdown In animals, when glucose is not

needed as a source of metabolic energy, it is stored, predomi-

nantly in liver and muscle cells, as glycogen, an ␣(1 S 4)-linked

glucan with ␣(1 S 6) branches every 8 to 14 residues. Glycogen

breakdown to glucose-6-phosphate (G6P) is a two-step process.

Glycogen phosphorylase catalyzes the phosphorolysis of the gly-

cosidic linkage of a terminal glucosyl residue to form glucose-1-

phosphate (G1P). Phosphoglucomutase interconverts G1P and

G6P. Glycogen debranching enzyme allows complete degrada-

tion of glycogen by catalyzing the transfer of three-residue chains

onto the nonreducing ends of other chains and catalyzing the hy-

drolysis of the remaining ␣(1 S 6)-linked glucosyl unit to glucose.

2 Glycogen Synthesis Glycogen is synthesized from

G6P by a pathway different from that of glycogen breakdown.

G6P is converted to G1P under the influence of phosphoglu-

comutase. UDP–glucose pyrophosphorylase utilizes UTP to

convert G1P to UDP–glucose, the activated intermediate in

glycogen synthesis. The hydrolysis of the PP

product by inor-

ganic pyrophosphatase drives the reaction to completion. Glu-

cosyl units are transferred from UDP–glucose to the C4 OH

group of a terminal residue on a growing glycogen chain by

glycogen synthase. The chain is initiated on glycogenin.

Branching occurs through the action of glycogen branching

enzyme, which transfers

⬃7-residue segments of ␣(1 S 4)-

linked chains to the C6 OH group of a glucosyl residue on

the same or another glycogen chain.

3 Control of Glycogen Metabolism The rates at which

glycogen is synthesized by glycogen synthase and degraded by

glycogen phosphorylase are controlled by the levels of their al-

losteric effectors such as ATP,AMP,G6P, and glucose. Superim-

posed on this allosteric control is control by the phosphoryla-

tion/dephosphorylation of these enzymes. The kinases and

phosphatases that catalyze these modifications are part of am-

plifying cascades that are ultimately controlled by the hor-

mones glucagon, insulin, and epinephrine, and by Ca

2⫹

Glucagon and epinephrine stimulate glycogen breakdown by

stimulating adenylate cyclase to increase the intracellular

[cAMP]. cAMP is a “second messenger” that activates protein

kinase A (PKA), which, through its activation of phosphory-

lase kinase, results in the phosphorylation of both glycogen

phosphorylase and glycogen synthase. Phosphorylation activates

glycogen phosphorylase but inactivates glycogen synthase. In ad-

dition, epinephrine causes an increase in the concentrations of

other second messengers, namely, inositol-1,4,5-trisphosphate

(IP

), diacylglycerol (DAG), and Ca

2⫹

, that reinforce the cAMP-

dependent responses. Ca

2⫹

, which is also released into muscle cy-

tosol by nerve impulses,binds to calmodulin so as to induce this

protein to activate protein kinases via an intrasteric mecha-

nism by which Ca

2⫹

–CaM extracts autoinhibitory sequences

from the kinases’ active sites.A decrease in [cAMP] and/or the

presence of insulin leads to the activation of phosphoprotein

phosphatase-1 to dephosphorylate glycogen phosphorylase

and glycogen synthase.

The phosphoproteins that participate in glycogen metabolism

are dephosphorylated through the action of phosphoprotein

phosphatase-1, which is only active when it is associated with a

glycogen particle through the intermediacy of glycogen-binding

or G

subunits. When [glucose] is high, the liver synthesizes

glucose-6-phosphate (G6P) and ultimately glycogen from glu-

cose via the action of glucokinase (GK), which has kinetic prop-

erties distinct from those of other hexokinases.When [glucose] is

low, GK is inhibited by glucokinase regulatory protein (GKRP)

and G6Pase hydrolyzes the G6P product of glycogen breakdown

(which is favored at low [glucose]) for export to other tissues.The

concentration in liver of F2,6P, an activator of PFK and an in-

hibitor of FBPase, is also dependent on the rates of cAMP-

dependent phosphorylation and dephosphorylation. It is both

synthesized and degraded through the action of PFK-2/FBPase-2,

whose enzymatic activities are oppositely controlled by both

allosteric regulation and phosphorylation/ dephosphorylation.

4 Glycogen Storage Diseases Glycogen storage dis-

eases are caused by a genetic deficiency of one or another of

the enzymes of glycogen metabolism. Ten different deficien-

cies of varying severity have been reported in humans.

668 Chapter 18. Glycogen Metabolism

CHAPTER SUMMARY

Type 0: Liver Glycogen Synthase Deficiency

The activity of liver glycogen synthase is extremely low

in individuals with this disease and hence they exhibit hy-

perglycemia after meals and hypoglycemia at other times.

However, the primary lesion may not always be in the syn-

thase itself because other metabolic defects may lead to an

imbalance of the glycogen synthase cyclic cascade.The root

cause of Type 0 glycogen storage disease is still under in-

vestigation. Muscle glycogen synthase deficiency disease

has recently been described. Its victims are easily fatigued

and develop heart abnormalities that may cause sudden

cardiac arrest. Note these are the only known glycogen

storage diseases in which there is a deficiency rather than

an overabundance of glycogen.This suggests that the com-

plete absence of glycogen is lethal.

Since many of the glycogen storage diseases have simi-

lar clinical symptoms, these diseases are best diagnosed via

genetic testing.

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670 Chapter 18. Glycogen Metabolism

PROBLEMS

1. A glycogen molecule consisting of 100,000 glucose residues

is branched, on average, every 10 residues with one branch per

tier. (a) How many reducing ends does it have? (b) How many

tiers of branches does it have, on average?

2. A mature glycogen particle typically has 12 tiers of

branches with 2 branches per tier and 13 residues per branch.How

many glucose residues are in such a particle?

3. Calculations based on the volume of a glucose residue and

the branching pattern of cellular glycogen indicate that a glycogen

molecule could have up to 28 branching tiers before becoming im-

possibly dense. What are the advantages of such a molecule and

why is it not found in vivo?

4. The free energy of hydrolysis of an (1 S 4) glycosidic

bond is 15.5 kJ ⴢ mol

1

, whereas that of an (1 S 6) glycosidic

bond is 7.1 kJ ⴢ mol

1

. Use these data to explain why glycogen

debranching includes three reactions [breaking and reforming

(1 S 4) bonds and hydrolyzing (1 S 6) bonds], while glycogen

branching requires only two reactions [breaking (1 S 4) bonds

and forming (1 S 6) bonds].

5. The complete metabolic oxidation of glucose to CO

and

yields 32 ATPs (Section 22-2Bc). What is the fractional ener-

getic cost of storing glucose as glycogen and later metabolizing the

glycogen rather than directly metabolizing the glucose? (Recall

that glycogen’s branched structure results in its degradation to

92% G1P and 8% glucose.)

6. What are the effects of the following on the rates of glyco-

gen synthesis and glycogen degradation: (a) increasing the Ca

2

concentration, (b) increasing the ATP concentration, (c) inhibit-

ing adenylate cyclase, (d) increasing the epinephrine concentra-

tion, and (e) increasing the AMP concentration?

7. Show that hexokinase activity but not glucokinase activity is

insensitive to blood [glucose] over the physiological range. Calcu-

late the ratio of glucokinase to hexokinase activities when [glu-

cose] is 2 mM (hypoglycemic), 5 mM (normal), and 25 mM (dia-

betic). Assume that K

 0.1 mM for hexokinase and that both

enzymes have the same V

max

8. Compare the properties of a bicyclic cascade with those of

a monocyclic cascade.

9. The V

max

of muscle glycogen phosphorylase is much larger

than that of liver. Discuss the functional significance of this

phenomenon.

10. How does epinephine act on muscles to prepare them for

“fight or flight”?

*11. A complication of glycogen metabolism that we have not

discussed is that many protein kinases, including phosphorylase

kinase, are autophosphorylating; that is,they can specifically phos-

phorylate and thereby activate themselves. Discuss how this phe-

nomenon affects glycogen metabolism, taking into consideration

the possibilities that phosphorylase kinase autophosphorylation

may be an intramolecular or an intermolecular process.

12. Explain the symptoms of von Gierke’s disease.

13. A sample of glycogen from a patient with liver disease is

incubated with P

, normal glycogen phosphorylase, and normal

debranching enzyme. The ratio of glucose-1-phosphate to glucose

formed in this reaction mixture is 100. What is the patient’s most

likely enzymatic deficiency? What is the probable structure of the

patient’s glycogen?

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