Quadrant - I 4.1. GLYCOGEN METABOLISM

Code and Title of the Paper: F10NB Nutritional Biochemistry

Code and Title of the Module: F10NB04 Glycogen Metabolism and Hexose Monophosphate

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Name of the Content Writer: Dr. S. Sumathi

Quadrant - I

4.1. GLYCOGEN METABOLISM

The major site of daily glucose consumption (75%) is the brain via

aerobic pathways. Most of the remainder of is used by erythrocytes, heart muscle, and skeletal muscle. The body gets glucose either directly

from the diet or from amino acids and lactate via gluconeogenesis. Glucose got from these two primary sources either remains soluble in

the body fluids or is stored in a polymeric form, glycogen. Glycogen is considered the main storage form of glucose and is found mostly in liver

and muscle. The kidney and intestines adds minor storage sites. With up to 10% of its weight as glycogen, the liver has the maximum specific

content of any body tissue. Muscle has a much lower quantity of glycogen per unit mass of tissue, but since the total mass of muscle is

so much greater than that of liver, total glycogen stored in muscle is about twice that of liver. Glycogen storage in the liver are considered

the main buffer of blood glucose levels.

OBJECTIVES

- To give an overview of glycogen metabolism

- To understand the factors that control the glycogen metabolism by intracellular signalling

- To understand the function of the pentose phosphate pathway in

production of NADPH and ribose precursors for nucleic acid synthesis.

Summary

Glycogen is the animal storage form of branched poly(glucose). The

storage polysaccharide of animals is glycogen. All cells contain glycogen, but it is most prevalent in the liver and the muscles. Glycogen comprises of glucose molecules linked together with α(1 →4)linkages with α(1 →6)

branch points occurring every 8 to 12 residues. The purpose of the high branched structure is to have many nonreducing ends so that glucose

can be rapidly mobilized in times of metabolic needs.Glycogen

homeostasis involves the concerted regulation of the rate of glycogen synthesis (glycogenesis) and the rate of glycogen breakdown

(glycogenolysis). These two processes are reciprocally regulated such that hormones that stimulate glycogenolysis (e.g. glucagon, cortisol,

epinephrine, norepinephrine) at the same time inhibit glycogenesis. Conversely, insulin, which directs the body to store excess carbon for



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future use, stimulates glycogenesis while concurrently inhibiting glycogenolysis.

Introduction

Glycogen metabolism is vital for several reasons.

• Glycogen stores in the liver are used to maintain a constant blood

glucose concentration. Glycogen storage is also maintained by muscles as

a reservoir of glucose for strenuous muscular activity.

• The synthesis and degradation of glycogen take place by different

metabolic pathways allowing for reciprocal regulation.

• In addition, the enzymes of glycogen metabolism are under hormonal

regulation.

The biochemical pioneers of glycogen metabolism were the Cori’s, Carl

and Gerty, a husband and wife team. They demonstrated that the

glycogen is broken down by phosphorolysis. The complete breakdown

process of glycogen breakdown is:

(glucose)n → glucose-1-phosphate + (glucose)n-1.

Processes of glycogen synthesis is:

(glucose)n-1 + UDP-glucose → (glucose)n

The glycogen breakdown and synthesis is controlled by two key enzyme

(glycogen phosphorylase and glycogen synthase) activities which are

activated/inactivated by allosteric regulation and phosphorylation /

dephosphorylation.

The storage form of glucose in most eukaryotic cells (except plants) is

glycogen, a large highly branched polysaccharide containing glucose units

joined by α-1→4 and α-1→6 glycosidic bonds. Both the liver and muscle

store glycogen and hence have the necessary anabolic and catabolic

enzymes. The degradation and synthesis of glycogen occurs in the cytosol

and the substrate for these reactions is the free ends of the branched

polymer. The large number of branch points in glycogen results in the



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generation of multiple nonreducing ends that provide a highly efficient

mechanism to quickly release and store glucose.

4.1.1.GLYCOGEN SYNTHESIS - Luis Leloir discovered the glycogen

biosynthetic pathway.

Glycogen + UDP-glucose → Glycogenn+1 + UDP

If we compare the synthetic pathway to the degradative pathway it is

clear that the glycogen biosynthesis is not merely the reversal of the

degradative pathway. The two pathways are distinct providing a

mechanism for reciprocal control.

A. UDP-glucose formation by UDP-glucose pyrophosphorylase - In the

glycogen synthesis pathway, at first, the uridine diphosphate(UDP) is

attached to glucose. The reaction is catalyzed by UDP-glucose

pyrophosphorylase.

B. Glycogen synthesis by glycogen synthase

1.The glycosidic bond between glucose and UDP in UDP-glucose is

hydrolyzed.

2. The glucose unit of UDP-glucose is transferred to the C4-OH group on

one of glycogen’s non-reducing ends to form an α (1→4) glycosidic bond.



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C. Glycogen Branching -

Glycogen synthetase can synthesize α(1→4) linkages. Another enzyme is

required to form the α(1→6) linkages that make the branches. The

branching enzymes takes a block of seven or so residues of a nonreducing

end and transfers these seven residues to an interior site and creates an

α(1→6) linkage. The chain that contributes the seven residues must be at

least 11 residues long, and the new branch point must be at least four

residues away from pre-existing branch points. About 7 units of the non-

reducing end of α-amylose chain are removed at α(1→4) linkage, and

reattached to the C6 of other α-amylose chain by α(1→6) linkage. This

transfer is carried out by amylo-(1,4→1→6)-transglycosylase (branching

enzyme).



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4.1.2.GLYCOGENOLYSIS

Three enzymes are required for glycogen breakdown

1. Glycogen phosphorylase



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(Glycogen)n + Pi ↔

(glycogen)n-1 + G1P (n residues).

Glycogen phosphorylase catalyzes this reaction. Glycogen phosphorylase

catalyzes the successive phosphorolysis of glucose residues from a

nonreducing end. The phosphorylated glucose cannot diffuse out of the

cell. Glycogen phosphorylase requires a pyridoxl-5’-phosphate cofactor.

The pyridoxl-5’-phosphate cofactor is covalently bound to a lysine residue

via a Schiff base.

Glycogen phosphorylase degrades glycogen’s α(1→4) glycosidic bonds

sequentially until it gets four residues away from a α(1→6) branch point

when its action ceases and another enzyme is required to remove the

branches.This enzyme is called the debranching enzyme. This enzyme

releases a glucose unit one by one until it reaches ~ five units (limit

branch) from a branch point. There are two forms of the enzyme

phosphorylase :Phosphorylase a → E-O-PO3 and

Phosphorylase b → No phosphate attachment.



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Reaction mechanism includes cleavage of the glycosidic bond by an acid

catalysis to form G1P. The other enzyme active site, the α-1,6-

glucosidase, cleaves the α(1→6) glycosidic linkage. After which the

glycogen debranching enzyme removes branches so that glycogen

phosphorylase can complete reaction. The glucose-1,6-bisphosphate

formed can dissociate out of the active site before transferring the C1

phosphoryl group. When this happens, the active site serine must be

rephosphorylated.

There is an enzyme called phosphoglucokinase which phosphorylates

glucose-1-phosphate to form glucose-1,6-bisphosphate which can bind to

the dephosphorylated enzyme and transfer the C1-phosphoryl group to

reactivate the enzyme and produce glucose-6-phosphate.

Phosphoglucomutase - G1P produced from the glycogen breakdown must

be converted to G6P in order to enter glycolysis or to produce glucose in

liver. Phosphoglucomutase catalyzes the conversion of G1P to G6P.

The reactions of glycogen biosynthesis are shown below.

1. Glucose-6-phosphate→ Glucose-1-phosphate ( Phosphoglucomutase)

2. Glucose-1-phosphate + UTP → UDP-Glucose + PPi (UDP-glucose

pyrophosphorylase)

3. PPi + H2O → 2Pi ( Inorganic pyrophosphatase)

4. UDP-Glucose + Glycogen UDP + Glycogen n+1 (Glycogen synthase)

5. UDP + ATP → UTP + ADP (Nucleotide diphosphokinase)

Sum: Glucose-6-phosphate + ATP + Glycogen + H2O → Glycogen n+1 +

ADP + 2Pi

90% of glycogen is phosphorylytically cleaved into glucose-1-phosphate

which is isomerized into glucose- 6-phosphate.10% are the branched

residues which are hydrolyzed into glucose which can be phosphorlated

into glucose-6-phosphate.The complete oxidation of glucose-6-phosphate

through glycolysis, the citric acid cycle and oxidative phosphorylation

yields 38 molecules of ATP. The overall efficiency of storage is 97%.

4.1.3. Regulation of glycogen metabolism



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Allosteric regulation of glycogen synthase and phosphorylase Hormonal control of glycogen metabolism

Regulatory differences between liver and muscle phosphorylase

Allosteric regulation of glycogen synthase

and phosphorylase

The allosteric regulatory effects exercised by glucose-6-phosphate, ATP

and AMP on glycogen phosphorylase and glycogen synthase make good physiological sense. Exhaustion of ATP is an excellent reason to release

glucose from the store in order to make some more. On the other hand, glucose-6-phosphate will be plentiful when glucose itself is abundant, and

therefore signals an opportunity for replenishing the glycogen stores.

2. Hormonal control of glycogen metabolism

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. Insulin is the anabolic hormone and promotes the storage of fuels. It

activates the storage of glucose as glycogen in the liver and muscles. The conversion of glucose into triacylglycerides and the storage of

triacylglycerides in the adipose tissue is also promoted by insulin. Glucagon is the hormone that promotes the mobilization of fuels.

Glucagon acts to maintain glucose availability in the absence of dietary glucose by stimulating the release of glucose from the liver. Glucogon

stimulates glycogenolysis and gluconeogenesis. Glucagon also activates

the mobilization of fatty acids from the adipose tissue. The sites of glucagon action are principally in the liver and adipose tissue. The release

of glucagon is suppressed by insulin and glucose. The lowest levels of glucagon occur after a high carbohydrate meal. All of glucagon’s effects

are opposed by the effects of insulin. The stimulation of insulin release suppresses the release of glucagon.



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The glycogen synthase and the phosphorylase respond in opposite ways to phosphorylation: The synthase is inactivated, whereas the

phosphorylase is activated.

3. Regulatory differences between liver and

muscle phosphorylase

Liver enzyme Muscle enzyme

Inhibition by glucose + −

Activation by Ca2+ − +

Activation by AMP even when

unphosphorylated

− +

There are regulatory differences between glycogen phosphorylase in muscle and liver. The liver enzyme inhibited by glucose. But this does not

inhibit the muscle enzyme. Ca2+ stimulates the muscle enzyme but not the liver enzyme. Recall that Ca2+ is also the trigger for muscle

contraction; the simultaneous stimulation of glycogen breakdown therefore anticipates an increased demand for ATP.

Inter organ relationships in glycogen metabolism

Liver glycogen utilization

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Muscle glycogen utilization The Cori cycle

The two tissues that have the most significant pools of glycogen are the liver and skeletal muscle. Liver glycogen is turned over quickly;

it serves as the major reserve of blood glucose during short-term fasts. After liver glycogen is depleted, muscle glycogen can be

drawn down; this, however, requires some roundabout metabolic trickery.

Liver glycogen utilization

The liver mobilizes glucose from its glycogen store via glycogen

phosphorylase and phosphoglucomutase, which yields glucose-6-phosphate. The latter is transported to the endoplasmic reticulum, where

it is dephosphorylated. Glucose is taken back to the cytosol and released into the bloodstream.Some of the glucose will be rephosphorylated before

making it out of the cell, creating the futile cycle.However, the dominant glucose phosphorylating enzyme in the liver is glucokinase. This enzyme

has fairly low affinity for glucose; therefore, enough glucose will escape

rephosphorylation and be released into the bloodstream.

Muscle glycogen utilization





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Muscle glycogen primarily serves the energy needs of muscle tissue

itself.During prolonged physical exercise, maximum of it is broken down to glucose-6-phosphate and then consumed via the usual pathways right

within the cells that stored it. As discussed above, this usage is facilitated

by calcium-mediated activation of glycogen phosphorylase.

Under suitable conditions, that is, prolonged fast without physical

exercise, muscle glycogen can also contribute to the replenishment of blood glucose. However, even though muscle cells have been shown to

express glucose-6-phosphatase and thus are, in principle, able to produce free glucose, they should find it difficult to release it. The reason is

muscle contains hexokinase, which has a much greater substrate affinity than glucokinase and therefore will keep the intracellular level of free

glucose well below the extracellular concentration. The net transport of glucose should therefore be directed inward at all times.



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The Cori cycle

While skeletal muscle relies on oxidative metabolism most of the time,

some other tissues, especially red blood cells and lymphocytes, which together depend mostly or even exclusively on anaerobic glycolysis even

under aerobic conditions. The peripheral tissues releases lactate and it is scooped up by the liver, which converts it back to glucose through

gluconeogenesis. The process is known as the Cori cycle, named after its discoverers Carl and Gerti Cori, who worked it out as early as 1929.

Skeletal muscles produce lactate at a very much higher rate during short bouts of maximal exercise when the ATP demand exceeds the capacity for

aerobic metabolism. During intense exercise, the cardiac blood output is

diverted from the visceral organs to skeletal muscle. Thus, when ATP demand exceeds the oxygen supply of skeletal muscle, the oxygen

shortfall would be even greater in the liver, should it indeed attempt to make enough ATP for sustaining the muscle through gluconeogenesis;

and even with adequate oxygen, its capacity for producing glucose would fall far short of the muscles’ voracious appetite.

4.2.HEXOSE MONOPHOSPHATE SHUNT

The hexose monophosphate pathway(Pentose phosphate pathway)is used

to produce the NADPH required for a variety of cellular functions. This

NADPH production is controlled by the relative levels of NADP and NADPH

that alter the activity of glucose-6-phosphate dehydrogenase. This is the

control enzyme for the pathway. The ribose-5- phosphate required for



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nucleotide and nucleic acid biosynthesisis also synthesized in the hexose

monophosphate pathway. All the enzymes after 6-phosphogluconate

dehydrogenase step in hexose monophosphate pathway are fully

reversible. These enzymes let the synthesis of ribose-5-phosphate from

the glycolytic intermediates fructose-6-phosphate and glyceraldehyde-3-

phosphate, and the conversion of ribose-5-phosphate into glycolytic

intermediates. NADPH is part of the mechanism that red blood cells use to

prevent oxygen-mediated damage to their membranes and proteins, and

to maintain haemoglobin in the oxidation state required for oxygen

transport.

4.2.1.Hexose monophosphate shunt

Use and Importance:

The hexose monophosphate pathway is used for production of NADPH

from NADP. The biosynthetic reactions such as cholesterol synthesis, fatty

acid synthesis, drug reduction requires NADPH.It also acts as a cofactor

for some non-synthetic enzymatic reactions.Furthermore, it is used for

the production of ribose for nucleotide and nucleic acid synthesis.

The hexose monophosphate pathway also permits the entry of some

carbohydrates into the glycolytic pathway (especially ribose, but also

some others), and hence acts as a connection route between different

pathways.

4.2.2.Overview of the shunt pathway

The HMP shunt pathway has oxidative and non oxidative phases. In

the oxidative phase, glucose-6-phosphate is oxidised with the generation

of 2 molecules of NADPH, and one molecule of pentose phosphate, with

the release of one molecule of CO2. In the non oxidative phase, the

pentose phosphate is transformed to intermediates of glycolysis.



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A. Oxidative phase

Step 1

Glucose-6-phosphate is oxidised by NADP+ to produce 6-phosphoglucono

lactone. In this reaction one molecule of NADPH is formed. Regulation is

effected by glucose-6- phosphate dehydrogenase enzyme.

Step 2

The lactone is hydrolysed by lactonase to form 6-phosphogluconate.

Step 3

This is an oxidative step together with decarboxylation.

The enzyme is 6- phosphogluconate dehydrogenase.

Here Ribulose 5 phosphate is formed and a second molecule ofNADPH is

generated.



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B. Non oxidative phase



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Step 4: Isomerisation The ribulose-5- phosphate is then isomerised to

ribose-5 phosphate or epimerised to Xylulose-5-phosphate.

Step 5: Transketolase reaction Transketolase is a thiamine

pyrophosphate (TPP) enzyme. This enzyme transfers two carbon unit with

keto group from Xylulose-5 phosphate to ribose-5-phosphate to a form 3

carbon (glyceraldehyde-3-phosphate) and 7 carbon (sedoheptulose-7

phosphate).

Step 6: Transaldolase reaction involves transfer of a 3 carbon unit

from sedoheptulose-7-phostate to glyceraldehyde-3-phosphate to form

fructose-6-phosphate.



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Step 7: second transketolase reaction In one more transketolase

reaction a 2C unit is transferred from Xylulose-5- phosphate to erythrose-

4-phosphate to form fructose-6- phosphate and glyceraldehyde-3-

phosphate.

Step 8: Regeneration of glucose -6- phosphate.

Two molecules of glyceraldehyde-3-phosphate formed in step 7 are

condensed to form one fructose-6-phosphate.This fructose-6-phosphate is

then converted to glucose-6-phosphate (reversal of step 2 of glycolysis).

4.2.3.SIGNIFICANCE

1.Pentose phosphate pathway protects cells against reactive oxygen

species (ROS)which includes molecular oxygen and partially reduced,

reactive forms of oxygen. Reduction of molecular O2 in a series of

one‐electron steps yields superoxide, hydrogen peroxide, hydroxyl radical,

and water which forms the reactive oxygen species (ROS)

2. NADPH and glutathione in protects the cells against highly reactive

oxygen derivatives. The cells are protected by reduced glutathione (GSH)

by destroying hydrogen peroxide and hydroxyl free radicals. The

regeneration of GSH from its oxidized form (GS‐SG) requires the NADPH

produced in the glucose 6‐ phosphate dehydrogenase reaction.

3.Importance in RBCs: The role of red blood cells in O2 transport makes

them liable for oxidative damage by H2O. H2O2 causes both oxidation of

iron in hemoglobin to form methemoglobin and lipid peroxidation in RBCs.

The production of NADPH is the major role of HMP in red blood cells that

protect these cells from oxidative damage by providing reduced

glutathione for removal of H2O2.



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4.Chemically NAD and NADP are very similar molecules. These two

molecules are structurally closely identical (the only difference is the

phosphate on the 2´-position of the adenosine ribose of NADP instead of

the free hydroxyl at this position in NAD). In catabolic processes (such as

the TCA cycle), NAD is used to accept electrons, whereas NADPH is

primarily used to donate electrons for synthetic reactions. NADPH can

thus act as a strong driving force for otherwise unfavorable reactions

because of both the concentration differential and the fact that NADPH

contains more energy than NADP. Variety of electron donation reactions

uses NADPH. In some cell types, NADPH is required to maintain normal

functioning in addition to its involvement in synthetic processes. As an

example, red blood cells, that perform very few synthetic reactions, yet

require significant amounts of NADPH. NADPH plays two critical roles in

red blood cells. These two roles are related to the oxygen-transport

function of the red blood cell. The first role is related to glutathione:

erythrocytes require NADPH to maintain their levels of reduced

glutathione.

5. Oxygen is toxic and without reduced glutathione, peroxides that are

spontaneously formed from molecular oxygen would oxidize the lipid

components of the red blood cell membranes. Furthermore, peroxides

have a tendency to damage hemoglobin, causing precipitation of the

protein. Insoluble aggregates of hemoglobin have severely impaired

oxygen carrying capacity, and insoluble protein aggregates also tend to

be inflexible enough to prevent the normal deformations of the red blood

cell. Two enzymes are essential to deal with the peroxides -Glutathione

peroxidase converts the peroxide to an alcohol using glutathione.

Glutathione reductase then uses NADPH to regenerate the reduced

glutathione. The second role of NADPH in red blood cells is associated to

hemoglobin. Oxygen tends to oxidize the hemoglobin iron from +2 to the

more stable +3 oxidation state (resulting in methemoglobin). This is

problematic: the +3 state of heme iron binds oxygen very poorly. NADPH

is used to supply reducing equivalents to methemoglobin reductase, the

enzyme which returns the haemoglobin to the +2 oxidation state.

6.Glucose-6-phosphate dehydrogenase deficiency:Glucose-6-phosphate

dehydrogenase deficiency is a very common genetic abnormality that is

found in 5-10% of the global population. The effect of the deficiency is a



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decreased ability to form NADPH in red blood cells. Malaria parasites live

inside red blood cells. The normal lifespan of red blood cells are about 120

days. The lifespan of the red blood cell is shortened by a number of

mutations (glucose-6-phosphate dehydrogenase deficiency, sickle cell

anaemia, and the thalassemias), causing death of the malaria parasites

that have not reached maturity. In glucose-6-phosphate dehydrogenase

deficiency, the most common mutations seem to decrease the half-life of

the enzyme that is effective in reducing the life-span of the cell since red

blood cells lack the ability to synthesize new proteins. The erythrocytes in

individuals with glucose-6-phosphate dehydrogenase deficiency are

asymptomatic unless challenged with oxidants; oxidant exposure,

however, results in hemolyticanemia (a reduced number of red blood cells

due to lysis of the cells).

4.2.4.Regulation of pentose phosphate pathway

• The entry of glucose 6‐phosphate into the pentose phosphate pathway

is controlled by the cellular concentration of NADPH. NADPH is a strong

inhibitor of glucose 6‐ phosphate dehydrogenase. As NADPH is used in

various pathways, inhibition is relieved, and the enzyme is accelerated to

produce more NADPH.The synthesis of glucose 6‐phosphate

dehydrogenase is induced by the increased insulin/glucagon ratio after a

high carbohydrate.

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Quadrant - I 4.1. GLYCOGEN METABOLISM