GLYCOGEN METABOLISM

Learning objectives:

Describe composition and glycosidic bonds in glycogen

Describe the biochemical pathway of glycogen synthesis

Describe the biochemical pathway of glycogenolysis

Discuss regulation of glycogen metabolism

Glycogen

Glycogen is a branched homopolysaccharide composed of α-D-glucose units bound by α-1,4 and (at branch points) α-1,6 glycosidic bonds.

On average, there are branches for every 8-10 glycosyl residues.

….. ….

O

H OH

OH H

CH2OH

H

H H O

H OH

OH H

CH2OH

H

H H O

H OH

OH H

CH2

H

H H O

H OH

OH H

CH2OH

H

H H

O O OO

O

α-1,4 α-1,4 α-1,4

α-1,6

α-1,4

α-1,4

O

H OH

OH H

CH2OH

H

H H O

H OH

OH H

CH2OH

H

H H O

H OH

OH H

CH2

H

H H O

H OH

OH H

CH2OH

H

H H

O O OO

O

H OH

OH H

CH2 OH

H

H

H

O

H OH

OH H

CH2 OH

H

H

H

O

H OH

OH H

CH2 OH

H

H

H

O

H OH

OH H

CH2 OH

H

H

H

O

O

O

O

Glycogen

….. ….

O

H OH

OH H

CH2OH

H

H H O

H OH

OH H

CH2OH

H

H H O

H OH

OH H

CH2

H

H H O

H OH

OH H

CH2OH

H

H H

O O OO

O

α-1,4 α-1,4 α-1,4

α-1,6

α-1,4

α-1,4

O

H OH

OH H

CH2OH

H

H H O

H OH

OH H

CH2OH

H

H H O

H OH

OH H

CH2

H

H H O

H OH

OH H

CH2OH

H

H H

O O OO

O

H OH

OH H

CH2 OH

H

H

H

O

H OH

OH H

CH2 OH

H

H

H

O

H OH

OH H

CH2 OH

H

H

H

O

H OH

OH H

CH2 OH

H

H

H

O

O

O

O

A single molecule can have a molecular mass of up to 108 Da withmore than 500,000 glucosyl residues.

Glycogen forms intracellular glycogen granules in the cytoplasm.

Electron micrograph of a section of a liver cell showing glycogen deposits as accumulations of electron dense particles (arrows).

Glycosyl residue attached by an α-1,6 glycosidic bond

Glycosyl residue at anon-reducing end

Glycosyl units are attachedand mobilized from thereducing ends

Glycogen is an intracellular storage form of readily available glucose

Main stores of glycogen in the human body:Liver - Approximately 100 g or 10% of the fresh weightMuscle - Approximately 400 g or 1-2% of the fresh weight

Most other cells have small amounts of glycogen stored

Glycogen

Glucose 6-P

Glucose

Blood glucose

Glycogen

Glucose 6-PG6Pase

GLYCOLYSIS

LIVER MUSCLE

Sources of blood glucose after a meal

Meal

Glycogen

Gluconeogenesis

8 16 24 2 7 30

Hours Days

mM glucose

8

4

Glycogen synthesisGlycogenesis

Glycogen is synthesized from molecules of α-D-glucose.

Synthesis occurs in the cytosol

Synthesis requires energy ATP for phosphorylation of glucose UTP for generating an activated form of glucose: UDP-glucose

Glucose

Glucose 6-phosphate

Glucose 1-phosphate

UDP-glucose

Glycogenn+1

Glycogenn+1 with an additional branch

Hexokinase/Glucokinase

Phosphoglucomutase

UDP-glucose pyrophosphorylase

Glycogen synthase

Branching enzyme

ATP

ADP

UTP

PPi

Glycogenn

2 Pi + H2O

Pyrophosphatase

Glycogen synthesis - Glycogenesis

O

H OH

OH H

CH2OH

H

OH

H H

OH

+ ATP + ADP

GlucokinaseHexokinase

Same reaction, same enzymes, and same regulation as in glycolysisIrreversible

Glucose Glucose 6-phosphate

O

H OH

OH H

CH2OPO32-

H

OH

H H

OH

Hexokinase

Glucose 6-phosphate (low phosphofructokinase activity)

Glucokinase

High blood glucose (release from GKRP, High Km)

Insulin stimulates gene transcription (only in liver)

-

++

O

H OH

OH H

CH2OPO32-

H

OH

H H

OH

O

H OH

OH H

CH2OH

H

OH

H H

OPO32-

O

H OH

OH H

CH2OPO32-

H

OH

H H

OPO32-

Glucose 6-phosphate

Glucose 1-phosphate

Glucose 1,6-bisphosphate

OHSer

OPO32-

Ser

OPO32-

Ser

Phosphoglucomutase

O

H OH

OH H

CH2OH

H

OH

H H

OPO32-

Glucose 1-phosphate

O

H OH

OH H

CH2OH

H

OH

H H

O – P – O – P – O - uridine

UDP-glucose

O O

O- O-

+ O- – P - O – P – O – P – O - uridine

O O O

O- O- O-

UTP

O- – P – O – P – O-

O O

O- O-

Pyrophosphate (PPi)

+

UDP-glucose pyrophosphorylase

O- – P – O – P – O-

O O

O- O-

Pyrophosphate (PPi)

+ H2O 2 Pi

Pyrophosphatase

NB: Irreversible reaction

Glucose 1-phosphate + UTP UDP-glucose + PPi

PPi + H2O 2 Pi

Glucose 1-phosphate + UTP + H2O UDP-glucose + 2 Pi

The irreversible hydrolysis of pyrophosphate drives the synthesis ofUDP-glucose

O

H OH

OH H

CH2OH

H

OH

H H

O – P – O – P – O - uridine

UDP-glucose

O O

O- O-

+

O

H OH

OH H

CH2OH

H

H H

HOO - R

α-1,4

Glycogen (n residues)

O

H OH

OH H

CH2

H

H H O

H OH

OH H

CH2OH

H

H H

O

α-1,4

O - Rα-1,4 HO

O- – P – O – P – O - uridine

O O

O- O-

+

Glycogen (n+1 residues)UDP

Glycogen synthase

Priming of glycogen synthesis

Glycogen synthase can NOT add glucosyl residues to free glucoseor to oligosaccharides of less than 8 glucosyl residues

Priming is catalyzed by the protein GLYCOGENIN

The first glucosyl residue is attached in an O-glycosidic linkageto the hydroxyl group of tyrosine of Glycogenin itself

7 additional residues are attached by glycogenin

Glycogenin remains attached to the reducing end of the glycogen molecule

TyrHO

Glycogenin

Tyr O

Glycogenin

8 UDP-glucose +

Glycogenin

…

Non-reducing end Cleaveage of α-1,4 bond

…

α-1,6bondNon-reducing ends

“Branching enzyme”Amylo-α(1,4) → α(1,6)-transglucosidase

Stoichiometry

Glucose + ATP + UTP + H2O + Glycogenn →

Glycogenn+1 + ADP + UDP + 2 Pi

Degradation of glycogenGlycogenolysis

Occurs in cytoplasm

Major product is glucose 1-phosphate from breaking α-1,4 bondsMinor product is glucose from breaking α-1,6 bonds

Glucose 1-phosphate : Glucose ≈ 10:1

Glycogenn

Glycogenn-1

Glycogen with branch

Glucose Glycogen with one less branch

Pi

Glycogen synthesis - Glycogenesis

Glucose 1-phosphate

Glucose 6-phosphate

Glucose

Phosphoglucomutase

G6PaseH2O

Glycolysis

Glycogen phosphorylase

…

“Debranching enzyme”

H2OPi

O

H OH

OH H

CH2OH

H

H H O

H OH

OH H

CH2

H

H H O

H OH

OH H

CH2OH

H

H H

O O

α-1,4 α-1,4

O - RHO

O- – P – OH

O

O-

+

+

O

H OH

OH H

CH2OH

H

OH

H H

OPO32-

Glucose 1-phosphate Glycogen with n-1 residues

O

H OH

OH H

CH2

H

H H O

H OH

OH H

CH2OH

H

H H

O

α-1,4

O - RHO

Phosphate Glycogen with n residues

Glycogen phosphorylase

NH

C

NH Lys

OH

CH3

2-O3PO-CH2

Pyridoxal phosphate is a coenzyme for the phosphorylasereaction.

Pyridoxal phosphate is bound to a nitrogen of a lysylresidue of glycogen phosphorylase

The phosphate of pyridoxal phosphate exchanges protons with the phosphate reactant, which allows the reactant to donate aproton to the oxygen atom on carbon 4.

+

O

H OH

OH H

CH2OH

H

OH

H H

OPO32-

O

H OH

OH H

CH2OPO32-

H

OH

H H

OPO32-

Glucose 1-phosphate

Glucose 1,6-bisphosphate

OHSer

OPO32-

Ser

OPO32-

Ser

Phosphoglucomutase

O

H OH

OH H

CH2OPO32-

H

OH

H H

OH

Glucose 6-phosphate

O

H OH

OH H

CH2OPO32-

H

OH

H H

OH

Glucose 6-phosphate

O

H OH

OH H

CH2OH

H

OH

H H

OH

Glucose

+ H2O + Pi

Glucose-6-phosphatase

(G6Pase)

Same reaction as in gluconeogenesisOccurs in endoplasmic reticulum and involves a glucose 6-phosphatase transporter and a catalytic subunit

The catalytic subunit is regulated at the level of transcription

…

α-1,6bond

Glycogen phosphorylase stops when 4 glucosyl units remain on each chain from a branch point

b c

a’

b’

a

c’

d’

d e

…b ca d eb’ c’a’

α-1,6bond

d’

Oligo-α(1,4)→α(1,4)-glucan transferase(debranching enzyme)

Amylo-α(1,6)-glucosidase(debranching enzyme)

…b ca d eb’ c’a’d’ +Glucose

H2O

Approximate Stoichiometry

Glycogenn+11 + 10 Pi + H2O →

Glycogenn + 10 Glucose 6-phosphate + Glucose

Glycogen

Glucose 6-P

Glucose

Blood glucose

Glycogen

Glucose 6-PG6Pase

GLYCOLYSIS

LIVER MUSCLE

Regulation of glycogen metabolism

Skeletal muscleGlycogen must be broken down to provide ATP for contraction, when the muscle is rapidly contracting,or in anticipation of contractions in stress situations like fear or excitement.

In rapidly contracting muscle: Low [ATP], High [AMP] High [Ca++]

Stress: High [Epinephrine]

Glycogen stores are replenished when muscles areresting.

Resting state: Low [AMP], High [ATP]

Hormonal regulation of metabolismHormone Type Secreted by Secreted in response to

Insulin Protein Pancreatic beta cells High blood [glucose]

Glucagon Polypeptide Pancreatic alpha cells Low blood [glucose]

Epinephrine Catecholamine Adrenal medulla Stress(adrenalin) Nervous system Low blood [glucose]

Glucocorticoids Steroid hormone Adrenal cortex StressLow blood [glucose]

Glucagon is the most important hormone signalinglow blood glucose concentration, while epinephrine andglucocorticoids play secondary roles.

Regulation of glycogen metabolism

LiverGlycogen must be broken down to provide glucosefor maintaining blood glucose in fasting or for providingadditional glucose for skeletal muscles in stress situations.

Fasting: High [Glucagon]

Stress: High [Epinephrine]

Glycogen stores must be replenished in the fed state

Fed state: High [Insulin] High [Glucose]

Muscle

Glycogen

Glucose 6-phosphate

Glycogen

Glucose 6-phosphate

Glycogen

Glucose 6-phosphate

Glycogen

Glucose 6-phosphate

Rapidly contracting stateStress

Resting state and withabundant energy

Fasting stateStress

Fed state

Liver

Key regulatory enzyme of glycogen breakdown:Glycogen phosphorylase

Key regulatory enzyme of glycogen synthesis:Glycogen synthase

Glycogen phosphorylase is a dimer of identical subunits.Glycogen phosphorylase can exist in an active R (relaxed) and an inactive T (tense) state.

In the T state, the catalytic site is partly blocked

Red: active site

Yellow:Glycogen binding site

Red site:Allosteric site forAMP binding

Blue/green sites:Phosphorylation sites

Regulation by energy state.

-

+ AMP (binding favors the active R state)

ATP (binding favors the inactive T state)

Allosteric regulation of glycogen phosphorylase

Regulation by feedback inhibition.

- Glucose 6-phosphate (G6P) G6P concentration increases when G6P is generated faster than it can be further metabolized, e.g. by glycolysis

Regulation by high blood glucose

- Glucose (Only liver glycogen phosphorylase)In the fed state with a high blood glucose concentration,there is no need for the liver to secrete glucose

Regulation of glycogen phosphorylase by phosphorylation

P

PInactive ActiveT state R state

Glycogenphosphorylase b

Glycogenphosphorylase a

ATP ADP

Phosphorylase kinase

Protein phosphatase 1 (PP1)

H2OPi

Phosphorylation occurs in the fasted or stressed stateDephosphorylation is stimulated in the fed state

Ca++Ca++

Ca++ Ca++

Ca++Ca++

Ca++ Ca++P P

P P

P P

P P

PKA

PP1

PKA

PP1

Inactive Partly active Fully active

Phosphorylation occurs in the fasted or stressed state.Dephosphorylation is stimulated in the fed state.Ca++ binding occurs when the [Ca++] is high, e.g. during rapid muscle contractions

Phosphorylase kinase is regulated by phosphorylation and Ca++ binding

One subunit is the Ca++ -binding calmodulin

Ca++

Ca++

Inactive Partly active Fully active

Cellmembrane

Glucagon(low blood glucose)

Glucagon receptor(liver) +

Adenylyl cyclase

Epinephrine(stress, fear)

Epinephrine receptor(muscle and liver)

ATP cAMP

+

+

Inactive ActiveProtein kinase A Protein kinase A (PKA)

Inactive ActivePhosphorylase kinase Phosphorylase kinase

P

Inactive glycogen Active glycogenphosphorylase b phosphorylase a

P

ATP ADP

ATP ADP

Glycogenn Glycogenn-1

Pi Glucose 1-phosphate

ATP cAMP + PPi

Adenylyl cyclase

cAMP

H2OPhosphodiesterase

AMP

Receptor

Adenylylcyclase

GDP

GTP

beta and gamma subunitof G-protein

alpha subunitof G-protein

Glucagon receptors and epinephrine receptors areG-protein-coupled receptor

GDP

When hormone is no longer present, intrinsic GTP hydrolase activity of the G-protein alpha subunithydrolyzes GTP to GDP, the alpha subunit re-associates with the beta and gamma subunits, andstimulation of adenylyl cyclase ends. cAMP is converted to AMP by phosphodiesterase. Thus, in the absence of hormone, the cAMP concentration rapidly falls.

β β

α αInsulin

Insulin receptorIt functions as atyrosine kinasewhen insulinis bound

β β

α α

P

P

P

P

Insulin receptor substrate

Autophosphorylation

β β

α α

P

P

P

P

P

Activation ofmultiple signalingpathways

Activationof proteinphosphatases

Activationof proteinkinases

In general, the protein kinases activated by insulin have opposite biological effects, the protein kinases activated by insulin have opposite biological effectsfrom those activated by glucagonfrom those activated by glucagon

In general, the protein phosphatases activated by insulin dephosphorylate proteins, the protein phosphatases activated by insulin dephosphorylate proteinsthat are phosphorylated by glucagon-stimulated protein kinases, such as PKAthat are phosphorylated by glucagon-stimulated protein kinases, such as PKA

Regulation of glycogen synthase

Regulation by feed-forward mechanism.

Glucose 6-phosphate (G6P) G6P concentration increases at high glucose concentrations when G6P is generated faster than it can be further metabolized

+

NB: Reciprocal regulation of glycogen synthaseand glycogen phosphorylase by glucose 6-phosphate

Regulation of glycogen synthase by phosphorylation

P

PActive Inactive

ATP ADP

PKA and Glycogen synthase kinase

Protein phosphatase 1 (PP1)

H2OPi

Phosphorylation occurs in the fasted or stressed stateDephosphorylation is stimulated in the fed state

Reciprocal regulation of glycogen phosphorylase andglycogen synthase by phosphorylation

P

PActive Inactive

ATP ADP

H2OPi

P

PInactive ActiveT state R state

ATP ADP

H2OPi

P P

P P

ATP ADP

H2OPi

Fasting/stress(glucagon/epinephrine)

PKA

Fed state (insulin) PP1

+

+

Phosphorylasekinase

Glycogenphosphorylase

Glycogensynthase

Active

And it is even more complex..

Scaffolding proteins of different subtypes in liver andmuscle can bind the glycogen particle, PP1, glycogen phosphorylase, and glycogen synthase

Binding brings participants of glycogen metabolismtogether.

Regulation of PP1 is itself complex with variousinhibitors responding to the metabolic state ofthe organism.