BCH3120
General Intermediary Metabolism
Lectures 5 and 6:
Glycogen metabolism and
gluconeogenesis
Dr Céline Aguer
celineaguer@montfort.on.ca
Lecture 5: Glycogen metabolism
In this course, we will see:
 what is glycogen, how and where it is stored
 glycogen catabolism when glycogen comes from food
 catabolism of stored glycogen
 glycogen synthesis
 regulation of glycogen metabolism
Learning objectives:
At the end of this lecture, you will be able to:
 Identify 2 types of O-glycosidic bounds on a glycogen molecule
 Describe glycogen biosynthesis
 Differentiate metabolic glycogen breakdown and dietary glycogen digestion
 Explain the different steps of glycogenolysis
 Explain the controls of glycogen metabolism
Chapters of interest:
• Biochemistry, 4th ed. Matthews: Chapter 13
2
Glycogen
3
Glycogen: what is it?
 Glycogen is a glucose polymer
 Multibranched homopolysaccharide
Nonreducing end
H
O-glycosidic linkage
 The molecules of D-glucose are held
together by intramolecular O-glycosidic
bonds (α1 -> 4) and intermolecular Oglycosidic bonds (α1 -> 6)
ONLY ONE!
4
Glycogen: why?
 Glucose storage in cells
 But why is glucose not simply stored as glucose????

One molecule of glycogen has the same osmotic pressure as one
molecule of glucose.

The O-glycosidic bounds stabilise the glucose units by making them
less oxidable.

Negative concentration gradient of glucose between the interior
and the exterior of the cell, therefore facilitating glucose entry.
5
Glycogen: where ?
 Glycogen is stored in:
 Liver: glycogen for « public usage »
• In case of need (fasting), the liver
exports glucose resulting from glycogenolysis
towards other organs
• Liver stores = 1/3 of stored glycogen in body = 150 g,
1/8 of liver weight!
• But liver stores can be exhausted in less than 24h!
 Muscles: glycogen for « private usage »
• Muscles use glycogen during muscle contraction
• Muscle stores = 2/3 of stored glycogen in body = 300 g,
only 0,8% of total muscle weight
• Muscle stores can be exhausted in 1 to 2h of exercise
6
Glycogen: where ?
Micrograph of the liver
mitochondria
Glycogen
granules
Endoplasmic
reticulum
7
Glycogen metabolism: introduction
8
Glycogen metabolism: what is it?
 Catabolism of glycogen =
 Catabolism of endogenous glycogen (tissue glycogen) = degradation of
glycogen in glucose-1-phosphate= glycogenolysis
 Catabolism of diet glycogen (glycogen from foods) = degradation of
glycogen in glucose
 Synthesis of glycogen = glycogenesis
9
Glycogen metabolism: when ?
 Glycogen synthesis or degradation depends on:
 The nutritional state of the organism
 The need for energy
10
Food glycogen catabolism
11
Food glycogen and starch catabolism
a-amylase
digestion
glucose
O-glycosidic bonds
(α1 -> 4) are broken by
hydrolysis:
Enzymes: α-amylase from
saliva and the pancreas
(digestive fluids): hydrolysis
of intra-chains (α1->4) to
give maltose and α-dextrin
CH2OH
CH2OH
CH2
O
O
O
O
O
CH2OH
+H2O
CH2OH
O
O
O
Maltose
OH
H
CH2
O
+
HO
α-dextrin…
Modified from: svt.ac-dijon.fr
12
Food glycogen and starch catabolism
α-dextrin :
composed of at least 6 units
of glucose with (α1->4)
bonds and one (α1->6) bond
wikipedia
13
Food glycogen and starch digestion
 The maltose is then hydrolyzed by maltase
 The α-dextrin is hydrolyzed by α-dextrinase which cleaves
(α1->6) bonds
 The final product is glucose, which then enters the blood stream
maltase
Glycogen
or starch
Maltose
glucose
α-amylase
α-dextrinase
glucose
α-amylase
α-dextrin
Maltase
α-dextrinase
14
Tissue glycogen catabolism:
glycogenolysis
15
Glycogenolysis
Step 1:
 The O-glycosidiques (α1 -> 4) bonds are broken by phosphorolysis, from
nonreducing ends.
 Enzyme: glycogen phosphorylase (in liver, muscles)
 The reaction stops 4 units of glucose before a (α1->6) branch.
 Limiting reaction
CH2OH
CH2OH
CH2OH
O
O
O
O
O
P
O
O
Glycogen
phosphorylase
+
HO
O
OH
OH
+
CH2OH
O
H
O
P
O
OH
OH
Phosphate group (Pi)
Glucose-1-phosphate
16
Glycogenolysis
Step 1:
Glycogen
phosphorylase
Pi
CH2OH
O
glycogen
phosphorylase
P
Glucose-1-phosphate
O
 Glucose-1-phosphate is then isomerized in glucose-6-phosphate by
phosphoglucomutase (we will see this in step 5)
17
Glycogenolysis
Debranching
enzyme
Step 2:
 Transfert of 3 molecules of
glucose (trisaccharide group) of
the lateral chain to the nonreducing end of another chain
Debranching enzyme
 Enzyme: debranching
enzyme :
glycosyltransferase
Only one glucose is left on the
side chain, linked to another
glucose by (α1->6) bond.
18
Glycogenolysis
Step 3:
Debranching
enzyme
 Hydrolysis of the (α1->6) bond
 Enzyme: debranching
enzyme: (α1->6) glucosidase
H2O
Debranching enzyme
CH2OH
 Produces one molecule of
glucose per (α1->6) bond
O
Glucose
Muscle:
Phosphorylated by hexokinase to
give glucose-6-phosphate.
Liver:
exported to blood circulation
19
Glycogenolysis
Step 4:
glycogen
phosphorylase
 Phosphorolysis of left over
(α1->4) bonds
 Enzyme: glycogen
phosphorylase
Pi
Glycogen phosphorylase
CH2OH
 Produces multiple
molecules of glucose-1phosphate
O
P
Glucose-1-phosphate
O
20
Glycogenolysis
Step 5:
 Isomerisation of glucose-1-phosphate in glucose-6-phosphate
 Enzyme: phosphoglucomutase
P
CH2OH
O
Glucose
(liver)
CH2
O
O
phosphoglucomutase
P
Glucose-1-phosphate
O
Glucose-6-phosphate
Glycolysis
( muscle)
 In the muscle, glucose-6-phosphate directly enters glycolysis
21
Glycogenolysis
Step 6: only in the liver
 Hydrolysis of glucose-6-phosphate in glucose
 Enzyme: glucose-6-phosphatase
 The glucose is then exported to the blood circulation
P
O
CH2
CH2OH
O
O
Blood
circulation
Glucose-6-phosphatase
Glucose-6-phosphate
H2O
Pi
Glucose
22
Glycogenolysis: overview
Muscles = glycolysis
phosphoglucomutase
Glucose 6-Phosphate
Glucose 6-phosphatase
Glucose
Liver
23
Lets think about this a little…
The syndrome of Gierke is an absence of glucose-6-phosphatase in the liver.
In your opinion, what happens to glycogen metabolism in patients with this
syndrome ?
What will be the consequence of this disease on liver glycogen content?
24
Glycogenesis
25
Glycogenesis:an overview
 Substrates: glucose and glucose activated in UDP-glucose
 2 principal steps:
 Formation of linear chains ((α1->4) bonds)
 Formation of side chains ((α1->6) bonds)
 Where?
 In the liver and the muscles
26
Glycogenesis
 Step 1:
 Phosphorylation of glucose in glucose-6-phosphate
 Enzymes: glucokinase or hexokinase
 Irreversible reaction
 Consumes an ATP
P
CH2OH
O
Glucose
Glucokinase
Hexokinase
ATP
ADP
O
CH2
O
Glucose-6-phosphate
Do you remember the differences between glucokinase and hexokinase?
How these differences can explain the different regulation of glycogen synthesis in the
muscle and the liver?
27
Glycogenesis
 Step 2:
 Isomerisation of glucose-6-phosphate in glucose-1-phosphate
 Enzyme: phosphoglucomutase
P
O
CH2
CH2OH
O
phosphoglucomutase
O
P
Glucose-1-phosphate
O
Glucose-6-phosphate
28
Glycogenesis
 Step 3:
 Glucose-1-phosphate is activated in UDP-glucose by UTP
 Enzyme: UDP-glucose pyrophosphorylase
CH2OH
CH2OH
O
O
UDP-glucose pyrophosphorylase
P
Glucose-1-phosphate
O
P
O
P P
UTP
uridine
PPi
H2O
pyrophosphatase
P
P
uridine
UDP-Glucose
2 Pi
29
Glycogenesis
 Step 4:
 Synthesis of linear chains (α1->4) : glucose of UDPglucose is transferred to the nonreducing end of a
glycogen primer or a linear chain undergoing
elongation
 Enzyme: glycogen synthase
 Limiting step!
 Consumes an ATP
CH2OH
O
O P
P
CH2OH
uridine
CH2OH
O
O
UDP-Glucose
glycogen synthase
CH2
O
O
ATP
ADP
P
P
P
P
(glucose)n
uridine
UDP
P uridine
UTP
O
CH2OH
CH2OH
O
CH2OH
O
O
O
CH2
O
O
O
(glucose)n+1
30
Glycogenesis
 Step 5:
 Building side chains (α1->6) bonds: after linear chain formation of about 10
molecules of glucose, the first 6 glucose of the non-reducing ends are
detached and transferred onto a glucose closer to the reducing end with
the formation of an O-glucosidic (α1->6) bond
 Enzyme: branching enzyme
Branching enzyme
31
Glycogenesis
 Step 6:
 Extension of the glycogen molecule by elongation of the branches and
formation of new (α1->4) bonds by glycogen synthase
 Addition of new side branches (new (α1->6) bonds) by branching enzyme
 Energy cost is high, each new glucose unit added onto the glycogen
molecule costs 2 ATP!
Where is the ATP used?
32
Regulation of glycogen metabolism
33
Regulation and control of glycogen metabolism
 Glycogenolysis: glycogen phosphorylase
 Glycogenesis: glycogen synthase
34
Regulation of glycogen phosphorylase
 2 types of regulation:
 Allosteric
 Covalent: 2 forms:
• Phosphorylated: form a, active
• Dephosphorylated: form b, low activity
35
Allosteric regulation of glycogen phosphorylase
 Allosteric regulation:
 Liver: glucose is an inhibitor
 Muscle:
• AMP = activator
• ATP and glucose-6-phosphate = inhibitors
36
Covalent regulation of glycogen phosphorylase
+ Glucagon (liver)
P
P
37
Regulation of glycogen synthase
 2 types of regulation:
 Allosteric
 Covalent: 2 forms:
• Dephosphorylated: form a, active
• Phosphorylated: form b, low activity
38
Allosteric regulation of glycogen synthase
 Allosteric regulation:
 Muscle: glucose-6-phosphate activates GS
Why G6P and not glucose activates glycogen synthase in muscle?
39
Covalent regulation of glycogen synthase
+ Glucagon (liver)
P
40
Covalent regulation of glycogen synthase
activation
Insulin
inhibition
Glucose
Hexokinase / Glucokinase
P
P
P
IRS-1 P
P
Phosphoglucomutase
Glucose-1-phosphate
Akt
P
Glucose-6-phosphate
P
UDP-glucose phosphorylase
UDP-glucose
GSK3b
P
P
Glycogen synthase
Glycogen (n glucose)
UDP
Glycogen (n+1 glucose)
Aguer – BCH3520 – Cours V – 25/01/2015
41
P
P
P
42
Lecture 6: Gluconeogenesis
In this lecture, you will see:
 What is gluconeogenesis and its function
 The various steps of gluconeogenesis according to the initial substrate
Chapters of interest:
• Biochemistry 4th ed.: chapter 13
Learning objectives
At the end of this lecture, you will be able to:
 Explain what gluconeogenesis is for
 Describe the various pathways to gluconeogenesis according to the initial substrate
Gluconeogenesis: introduction
Gluconeogenesis: what is it?
Gluconeogenesis is the synthesis of new molecules of glucose or glycogen from
non-carbohydrate sources
Gluconeogenesis: why ?
Gluconeogenesis is necessary when needs in glucose are greater than what has
been eaten or stored.
Gluconeogenesis: where ?
Glycolysis occurs mainly in
the muscle and brain
Gluconeogenesis occurs
mainly in the liver (90%)
and in the kidneys and
intestine (10%)
Gluconeogenesis: when ?
 Almost always active, although slowed in the post-prandial period.
Generates 50 g of glucose a day in a normal diet


When fasting:

Activated during the first hours of fasting.

If fasting period is greater than 1.5 day, hepatic gluconeogenesis is the
only source of glucose. The liver can produce up to 100 g of glucose a day
via this pathway.

If fasting carries on, hepatic gluconeogenesis diminishes and renal
gluconeogenesis progressively augments, after about 10 days, both
systems are equally generating glucose.
During anaerobic muscular activity:

Lactate is the main source of hepatic gluconeogenesis
Gluconeogenesis: with what?
gluconeogenesis precursors Importance
Origin
Pyruvate and lactate
1/3 of precursors
Red blood cells, muscles
Alanine
1/3 of precursors
Muscles
Glycerol
1/12 of precursors
Adipose tissue, food lipid
catabolism and lipoproteins
Glucogenic amino acids
1/8 of precursors
Food or tissue proteins
Propionate
1/8 of precursors
FA catabolism
Gluconeogenesis: how ?
 Gluconeogenesis is pretty much the reverse of glycolysis, EXCEPT for the 3
irreversible steps of glycolysis
Glycolysis
Glucose
Hexokinase, glucokinase
Glucose-6-phosphate
Fructose-6-phosphate
phosphosfructokinase
Fructose-1,6-bisphosphate
Glyceraldehyde-3
-phosphate
Dihydroxyacetone
phosphate
Gluconeogenesis
Glucose
Glucose-6-phosphatase
Glucose-6-phosphate
Fructose-6-phosphate
Fructose-1,6
-bisphosphatase
Fructose-1,6-bisphosphate
Dihydroxyacetone
Glyceraldehyde-3
phosphate
-phosphate
glycerol
Phosphoenolpyruvate
Phosphoenolpyruvate
Pyruvate kinase
pyruvate
Oxaloacetate
pyruvate
Alanin, lactate,
Glucogenic amino acids
mitochondrion
oxaloacetate
Malate
Krebs
cycle
50
Gluconeogenesis: how ?
Gluconeogenesis
Glucose
Glucose-6-phosphatase
Glucose-6-phosphate

The 3 gateways to gluconeogenesis :
 pyruvate
 phosphoenolpyruvate
 dixydroxyacetone phosphate
Fructose-6-phosphate
Fructose-1,6
-biphosphatase
Fructose-1,6-biphosphate
Dihydroxyacetone
Glyceraldehyde-3
phosphate
-phosphate
glycerol
Phosphoénolpyruvate
Oxaloacetate
Malate
pyruvate
Krebs
cycle
Alanine, lactate,
Glucogenic AA
mitochondria
Glucogenic AA
Aguer – TMM3503 – CoursVI – 06/02/2018
51
Gluconeogenesis from pyruvate
Gluconeogenesis from pyruvate

Step 1:

carboxylation of pyruvate in oxaloacetate

Enzyme: pyruvate carboxylase

Where? Mitochondrial matrix

1/3 of gluconeogenesis precursors come from pyruvate!
CH3
C
COOH
O
Pyruvate
Pyruvate
carboxylase
CO2 ATP
HOOC
ADP + Pi
CH2
C
COOH
O
Oxaloacetate
mitochondrion
Gluconeogenesis from pyruvate

Step 2:

Reduction of oxaloacetate in malate
 Enzyme: malate dehydrogenase with
coenzyme NADH,H+

HOOC
CH2
CH
COOH
OH
malate
Malate
dehydrogenase
NAD+
NADH,H+
Where? Mitochondrial matrix
HOOC
CH2
C
O
oxaloacetate
COOH
Gluconeogenesis from pyruvate
HOOC

Step 3:

malate exits the mitochondrion

Oxidation of malate in oxaloacetate

Enzyme: malate dehydrogenase

Where? In the cytosol
CH2
CH
COOH
OH
malate
Malate
dehydrogenase
HOOC
CH2
NAD+
NADH,H+
C
O
oxaloacetate
COOH
Gluconeogenesis from pyruvate
The malate shuttle
Gluconeogenesis from pyruvate

Step 4:
HOOC

Where? In the cytosol
C
COOH
O
oxaloacetate
 Phosphorylating decarboxylation of
oxaloacetate in phosphoenolpyruvate
 Enzyme: phosphoenolpyruvate
carboxykinase
CH2
Phosphoenolpyruvate
carboxykinase
CH2
C
GTP
GDP
CO2
COOH
O
PP
phosphoenolpyruvate
ADP
ATP
Gluconeogenesis from pyruvate
Gluconeogenesis from pyruvate
 Steps 5-10:
 Reverse reactions
of glycolysis
phosphoenolpuruvate
Enolase
5
2-phosphoglycerate
Phosphoglycerate
6
mutase
3-phosphoglycerate
Phosphoglycerate
ATP
7
kinase
ADP
1,3-bisphosphoglycerate
Glyceraldehyde-3-phosphate
NADH,H+ 8
dehydrogenase
NAD+, Pi
Glyceraldehyde-3 9
Dihydroxyacetone
-phosphate
phosphate
Triose phosphate isomerase
Aldolase
10
Fructose-1,6-biphosphate
Gluconeogenesis from pyruvate
Gluconeogenesis from pyruvate

Step 11:
 Hydrolysis of fructose-1,6bisphosphate into fructose-6-phosphate
O CH2 O CH2
P
O
P
Fructose-1,6-bisphosphate
H2O
 Enzyme: fructose-1,6bisphosphatase
Pi
P
Fructose-1,6bisphosphatase
O CH2 O CH2OH
Fructose-6-phosphate
What is the enzyme that catalyses the inverse reaction in glycolysis?
Gluconeogenesis from pyruvate
P

Step 12:

Reverse reaction of step 2 of glycolysis
O CH2 O CH2OH
Fructose-6-phosphate
Phosphohexose
isomerase
 Isomerisation of Fructose-6-phosphate
in Glucose-6-Phosphate.
P
O CH2
O
Glucose-6-phosphate
Gluconeogenesis from pyruvate
P

Step 13:

Hydrolysis of G6P into glucose

Enzyme: glucose-6-phosphatase
 Enzyme present within the endoplasmic
reticulum of the liver and kidneys
O CH2
O
Glucose-6-phosphate
H2O
Glucose-6phosphatase
Pi
HOCH2
O
Glucose
Energy payoff
What is the energy payoff of gluconeogenesis from pyruvate?
Glucose
Glucose-6-phosphate
Fructose-6-phosphate
Fructose-1,6-bisphosphate
Dihydroxyacétone
Glycéraldéhyde-3-phosphate
phosphate
1,3-bisphosphoglycérate
ATP
3-phosphoglycérate
ATP
2-phosphoglycérate
Phosphoénolpyruvate
Oxaloacétate
x2
pyruvate
ATP
oxaloacétate
Malate
Aguer – TMM3503 – CoursVI – 06/02/2018
64
Gluconeogenesis from lactate
Gluconeogenesis from lactate

In muscles:
 During anaerobic glycolysis, muscles produce lactate (see lecture 2)
 This lactate is exported outside of the muscles into the liver. In the liver,
lactate is transformed back into glucose re-exported back into the
muscles and either used immediately or stored as glycogen.
= CORI CYCLE
+
NADH,H+ NAD
CH3
C
COOH
CH3 CH COOH
O
Lactate dehydrogenase OH
pyruvate
lactate
MUSCLE
+
NAD+ NADH,H
CH3
CH COOH
OH
lactate
LDH
LIVER
Gluconeogenesis from alanine
Gluconeogenesis from alanine

Alanine is produced during the catabolism of amino acids in muscles

Alanine is produced by a double transamination:
1) transfer of amino group onto α-ketoglutarate
Enzyme: aminotransferase
2) transfer of amino group onto pyruvate
Enzyme: alanine aminotransferase
Amino acids
Α-ketoglutarate
Aminotransferase
α-ketonic acid
Glutamate
 alanine is converted to pyruvate in the liver
 This pyruvate enters gluconeogenesis
= FELIG CYCLE
Alanine
Alanine
aminotransferase
Pyruvate
To the liver
Gluconeogenesis from glycerol
Gluconeogenesis from glycerol
Where does glycerol come from? Triglyceride hydrolysis
In the liver and kidneys only:

Glycerol is phosphorylated in glycerol-3-phosphate
 Enzyme: glycerol kinase

Glycerol-3-phosphate is oxidized in dihydroxyacetone phosphate
 Enzyme: glycerol-3-phosphate dehydrogenase with coenzyme NAD+
ATP
CH2
CH CH2
OH
OH OH
glycerol
NAD+ NADH,H+
ADP
P
Glycerol
kinase
In liver and
kidneys only
O CH2
CH CH2
P
O CH2
C
CH2
OH OH Glycerol-3-phosphate
O
OH
dehydrogenase
glycerol-3Dihydroxyacetone
phosphate
phosphate
+ Glyceraldehyde3-phosphate
F-1,6biP
Glucose
Gluconeogenesis from glucogenic
amino acids
Gluconeogenesis from glucogenic amino acids

All amino acids, except leucine and lysine, are glucogenic
Gluconeogenesis
PEP
Alanine
Threonine
Cystein
Serine
Pyruvate
Oxaloacetate
Oxaloacetate
Asparagine
Aconitate
Cycle de Krebs
Tyrosine
mitochondrion
Citrate
Malate
Phenylalanine
Glycine
Acetyl-CoA
malate-aspartate
shuttle
Aspartate
Tryptophane
Fumarate
Succinate
FA with an odd
number of
carbons
α-ketoglutarate
Histidine
Proline
Arginine
Glutamate
Succinyl-CoA
Propionyl-CoA
Valine
Isoleucine
Methionine
Threonine
Glutamine
Next lecture…
Dr. Dave Patten: Mitochondrial supercomplexes
QUIZ (on lectures 4, 5 and 6)