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Transcript
Cholesterol Metabolism
Dr Nancy Carmichael
Thursday 22nd November 2007
1
Objectives
•
•
•
•
•
Roles of cholesterol in the body
Basic structure of cholesterol
Stages in the synthesis of cholesterol
Regulation of cholesterol synthesis
Cholesterol as a precursor for bile salts and
steroid hormones
• Cholesterol transport and the role of LDL
• Diseases related to cholesterol metabolism
and their treatment
2
Cholesterol
• Very important steroid
• Has roles in modulating the fluidity of animal
cell membranes and is the precursor of steroid
hormones such as progesterone, testosterone,
oestradiol, and cortisol
• It can be consumed in the diet or synthesised
de novo
• Synthesis and utilization of cholesterol must be
tightly regulated in order to prevent overaccumulation and abnormal deposition within
the body
• Accumulation of cholesterol can lead to
atherosclerosis, a disease of the coronary
3
arteries
Structure of Cholesterol
4
Cholesterol in the Lipid Bilayer
• Eukaryotic plasma
membrane has large
amounts of cholesterol
– up to 1 molecule for
every phospholipid
molecule
• Fills space between
phospholipid
molecules next to each
other
• Makes bilayer stiffer,
less fluid, and also less
permeable
5
Origin of Carbon Atoms in Cholesterol
•
All 27 carbon atoms in cholesterol come
from acetate
1. Label acetate – feed to rats. Cholesterol
synthesised by rats contained the label
2. Label acetate on either the methyl or carboxyl
carbon
6
Cholesterol Carbon Numbering
7
Synthesis of Cholesterol
1. Stage one is the synthesis of isopentenyl
pyrophosphate, an activated isoprene unit that
is the key building block of cholesterol.
2. Stage two is the condensation of six molecules
of isopentenyl pyrophosphate to form
squalene.
3. In stage three, squalene cyclises and is
converted to cholesterol
8
9
Stage 1: Synthesis of Isopentenyl
Pyrophosphate
i. First, 2 acetyl-CoAs form acetoacetyl-CoA
ii. Then acetoacetyl-CoA and acetyl-CoA
combine to make 3-hydroxy-3methylglutaryl CoA (3-HMG CoA)
iii. 3-HMG CoA is then reduced to
mevalonate in the cytosol
iv. Mevalonate is converted to isopentenyl
pyrophosphate
10
(i) 2 acetyl-CoAs form acetoacetylCoA
11
(ii) Formation of 3-HMG CoA
12
(iii) 3-HMG CoA to Mevalonate
• 3-HMG CoA has 2 fates:
– Conversion to mevalonate for synthesis of
cholesterol – cytosol
– Cleaved to form acetyl CoA and acetoacetate mitochondria
• The synthesis of mevalonate is the
committed step in cholesterol formation
• The enzyme catalysing this irreversible step,
(3-HMG-CoA reductase), is an important
control site in cholesterol biosynthesis
13
14
(iv) Mevalonate to Isopentenyl
Pyrophosphate
• Mevalonate is converted into 3-isopentenyl
pyrophosphate in:
– three consecutive reactions requiring
ATP
– a decarboxylation reaction
15
(iv) Mevalonate to Isopentenyl Pyrophosphate
16
(iv) Mevalonate to Isopentenyl Pyrophosphate
17
(iv) Mevalonate to Isopentenyl Pyrophosphate
18
(iv) Mevalonate to Isopentenyl Pyrophosphate
19
Stage 2: Isopentenyl
Pyrophosphate to Squalene
• Squalene is synthesised from isopentenyl
pyrophosphate in reactions involving the following
number of carbon atoms:
C5 → C10 → C15 → C30
i. isomerisation of isopentenyl pyrophosphate (C5) to
dimethylallyl pyrophosphate (C5)
ii. isopentenyl pyrophosphate and dimethylallyl
pyrophosphate condense to form a geranyl
pyrophosphate (C10) (enzyme; geranyl transferase)
iii. Geranyl pyrophosphate then combines with
isopentenyl pyrophosphate to form farnesyl
pyrophosphate (C15) (enzyme: geranyl transferase)
iv. two molecules of farnesyl pyrophosphate combine to
form squalene (C30) (enzyme: squalene synthase) 20
(i) Isomerisation of Isopentenyl
Pyrophosphate to Dimethylallyl
Pyrophosphate
21
(ii) Formation of Geranyl
Pyrophosphate
22
(iii) Formation of Farnesyl Pyrophosphate
23
(iv) Formation of Squalene
24
Stage 3: Squalene forms Cholesterol
i.
First stage, squalene to squalene epoxide, is
a reduction reaction requiring O2
ii. Squalene epoxide is cyclised to lanosterol
by a cyclase enzyme
•
•
Migration of 2 methyl groups
movement of electrons through 4 double bonds
iii. Lanosterol is converted to cholesterol by:
•
•
•
removal of 3 methyl groups
reduction of a double bond by NADPH
migration of another double bond
25
(i) Squalene to
Squalene
Epoxide
26
(ii) Squalene
Epoxide to
Lanosterol
• Migration of 2
methyl groups
• Movement of
electrons
through 4
double bonds
27
(iii) Lanosterol to Cholesterol
HCOOH + 2CO2
• Removal of 3
methyl groups
• Reduction of
one double
bond by
NADPH
• Migration of
another
double bond
28
Summary of Cholesterol Biosynthesis
29
Regulation of Cholesterol Synthesis
• Cholesterol can be obtained from the diet or it can
be synthesised de novo
• The liver is the major site of cholesterol synthesis in
mammals, although the intestine also forms
significant amounts
• The rate of cholesterol formation by these organs is
very responsive to the cellular level of cholesterol
• This is called feedback regulation
• Here, it is mediated mostly by changes in the
amount and activity of 3-HMG CoA reductase
• 3-HMG-CoA reductase is controlled in many ways
30
HMG CoA Reductase Regulation
1. Rate of synthesis of 3-HMG CoA
reductase mRNA
2. Rate of translation of 3-HMG CoA
reductase mRNA into protein
3. Degradation of 3-HMG CoA reductase
4. Phosphorylation of 3-HMG CoA
reductase
31
HMG CoA Reductase Regulation
DNA
1
mRNA
2
protein
3
degradation
products
4
protein P
32
1.Synthesis of HMG CoA
Reductase mRNA
• The rate of synthesis of reductase mRNA
is controlled by the sterol regulatory
element binding protein (SREBP)
• This transcription factor binds to a short
DNA sequence called the sterol
regulatory element (SRE) on the 5’ side
of the reductase gene
• In the presence of sterols, SRE inhibits
mRNA production
33
2. Translation of HMG CoA
Reductase mRNA
• The rate of translation of reductase mRNA
is inhibited by nonsterol metabolites
derived from mevalonate, as well as by
dietary cholesterol.
34
3. Degradation of HMG CoA
Reductase
• The enzyme is bipartite:
– cytosolic domain carries out catalysis
– membrane domain senses levels of
derivatives of cholesterol and
mevalonate
• A high level of these products leads to
rapid degradation of the enzyme
35
4. Phosphorylation of HMG CoA
Reductase
• Phosphorylation decreases the activity of the
reductase
• Hormones regulate phosphorylation:
– Glucagon stimulates phosphorylation
(deactivation)
– Insulin stimulates dephosphorylation
(activation)
36
Negative Feedback of Cholesterol
Synthesis
• All four regulatory mechanisms are
modulated by receptors that sense the
presence of cholesterol in the blood
• Negative feedback inhibition
37
Fates of Cholesterol
• Most cholesterol synthesis takes place in liver
• Some of this is incorporated into membrane of liver
cells
• Most is exported in the forms of:
– Bile acids and their salts
– Cholesteryl esters
• Cells use the cholesterol for membrane synthesis
• They can also use it as a precursor for steroid
hormone production and vitamin D production
38
Bile Acids and Salts
• Bile acids and salts are derivatives of cholesterol
• Make good detergents as they contain polar and
non-polar regions
• They are synthesised in the liver
• They are the main constituent of bile
• They emulsify dietary lipids, which increases their
surface area to:
– Promote hydrolysis by lipases
– Facilitates their absorption by the intestine
• Bile salts also aid in absorption of lipid soluble
vitamins
39
Bile Salts as an Emulsifier
40
Excretion of Cholesterol in Bile
• Synthesis of bile acids is one of the
predominant mechanisms for the excretion
of excess cholesterol
• However, the excretion of cholesterol in
the form of bile acids is insufficient to
compensate for an excess dietary intake
of cholesterol.
41
Synthesis of Bile Acids
• Bile acids are synthesised from cholesterol
via many reactions
• The first reaction, cholesterol to 7ahydroxycholesterol (enzyme: 7a-hydroxylase)
is the rate limiting step in bile acid synthesis
• Conversion of 7a-hydroxycholesterol to the
bile acids requires several steps (not shown in
diagram)
42
43
Bile Acids
• The most abundant bile acids in human bile
are chenodeoxycholic acid and cholic acid
• These are the primary bile acids
• In intestines, primary bile acids are
converted to the secondary bile acids
– deoxycholate (from cholate)
– lithocholate (from chenodeoxycholate)
44
Synthesis of Bile Salts
• In the liver the carboxyl group of primary
and secondary bile acids is conjugated via
an amide bond to either glycine or taurine
• React with glycine to form glycocholate
• React with taurine (a derivative of cysteine:
H2N-CH2-CH2-SO3-) to form taurocholate
• Glycocholate is the main bile salt
• They are secreted into the intestine, where
they aid in the emulsification of dietary
lipids
45
Synthesis of Bile Salts
• Glycocholiate (glycocholic acid)
• Taurocholate (taurocholic acid)
46
Cholesterol used to Synthesise Steroids
• Cholesterol is the precursor of the five major
classes of steroid hormones:
– progestagens
– glucocorticoids
– mineralocorticoids
– androgens
– oestrogens
• These hormones are powerful signal
molecules that regulate many processes in
the body
47
Cholesterol and Steroid Hormones
Cholesterol (C27)
Pregnelonone (C21)
Progestagens (C21)
Mineralocorticoids
Androgens (C19)
(C21)
Glucocorticoids (C21)
Oestrogens (C
48 19)
Cholesteryl Esters
• About 2/3 of cholesterol in blood is in form of an
ester
• Cholesteryl esters are formed in the liver via the
action of acyl-CoA-cholesterol acyl transferase
(ACAT)
• Catalyses transfer of a fatty acid from coenzyme A
to the hydroxyl group of cholesterol
• This changes cholesterol into a more hydrophobic
form
• Can be stored in the liver or transported to other
tissues which need cholesterol
49
50
Transport of Cholesterol in the Blood
•
•
•
•
•
Cholesterol and cholesteryl esters are
virtually insoluble in water
They need to be transported from origin to
other tissues for storage or use
They are transported in body fluids in the
form of lipoprotein particles
These are complexes of an apolipoprotein
combined with cholesterol, cholesteryl
esters, phospholipids or triacylglycerol
Apolipoprotein is the protein in its lipid free
51
form
Transport of Cholesterol in the Blood
•
•
Different combination of apolipoproteins
and lipids produce particles of different
density
Lipoproteins are classified according to
increasing density
–
–
–
–
–
–
Chylomicrons
Chylomicron remnants
Very low density lipoproteins (VLDL)
Intermediate-density lipoproteins (IDL)
Low density lipoproteins (LDL)
High density lipoproteins (HLDL)
52
Low
Density
Lipoprotein
53
54
Specific Function of Lipoproteins
• Each class of lipoproteins has a specific
function
• This depends on its point of synthesis, lipid
composition and apolipoprotein content
• At least 9 apolipoproteins are found in
lipoproteins of human plasma
• The apolipoprotein has 2 roles:
1.Solubilise hydrophobic lipids
2.Contain targeting signals – target
lipoprotein to a specific tissue or enzyme 55
Chylomicrons
• Largest, least dense, contain many triacylglycerols
• Synthesised in endoplasmic reticulum of epithelial
cells in small intestine, then enter blood stream
• Carry fatty acids to where they are used or stored
• The chylomicron remnants (without fats but still
containing cholesterol) move to liver
• apoE in the chylomicron remnants bind receptors
in liver – taken up by endocytosis
• In liver, they release cholesterol and are degraded
in lysosomes
56
VLDL
• If diet has more fatty acids than needed – converted to
triacylglycerols in liver and packaged into VLDL
• Excess carbohydrate can also be dealt with in the same
way
• VLDLs are transported in the blood from liver to muscle
and adipose tissue
• Here the fatty acids are relased from the triacylglycerols
in VLDL
• Adipocytes take up the fatty acids and resynthesise
triacylglycerols to store
• Most VLDL remnants (IDL) removed from circulation by
hepatocytes: are uptaken by endocytosis (if apoE
present)
• Half the VLDL become LDL due to loss of lipids
57
58
Chylomicrons give
plasma a milky
appearance
59
LDL
• Loss of triacylglycerols converts some VLDL
to VLDL remnants (IDL) and then to LDL
• LDLs are very rich in cholsterol and
cholesteryl esters
• They have apoB-100 as the major
apolipoprotein
• They carry cholesterol to extrahepatic tissue
which has receptors to recognise apoB-100
• The receptors mediate the uptake of
cholesterol and cholesteryl esters
60
HDL
• These begin in liver and small intestine
• Contains very little cholesterol and no
cholesteryl esters
• Very protein rich
• Contain enzyme LCAT (lecithin-cholesterol
acyl transferase)
• LCAT catalyses formation of cholesteryl
esters from lecithin (phosphatidylcholine)
and cholesterol
61
Formation of Cholesteryl Esters from
Lecithin
62
HDL and LCAT
• LCAT is on the surface of nascent (newly
forming) HDL particles
• It converts the cholesterol and lecithin of the
chylomicron and VLDL remnants to
cholesteryl esters
• These form a core and makes HDL
molecules from disc shaped to spherical
• HDL particles are then mature
• This cholesterol-rich lipoprotein then returns
to the liver where it unloads some
cholesterol which is converted to bile salts 63
HDL
• HDL may be taken up by the liver by receptor
mediated endocytosis
• Some is taken to other tissues
• HDL binds to plasma membrane receptor
proteins (SR-BI) in hepatic and steroidogenic
tissues e.g. adrenal gland
• These mediate the selective transfer of
cholesterol and other lipids in HDL into the cell
• The depleted HDL then dissociates to recirculate in the blood stream and extract more
lipids from chylomicron and VLDL remnants 64
Reverse Cholesterol Transport
• Depleted HDL can also pick up cholesterol
stored in extrahepatic tissues and carry it to
the liver
• This is called reverse cholesterol transport
• In one reverse cholesterol transport pathway,
interaction of nascent HDL with SR-BI
receptors in cholesterol-rich cells triggers
passive movement of cholesterol from the
cell surface into HDL
• HDL then carries it back to liver
65
Reverse Cholesterol Transport
• Another reverse cholesterol transport
pathway – apoA-I in depleted HDL interacts
with an active transporter in a cholesterolrich cell
• This is the ABC1 protein
• The apoA-I and the HDL are taken up by
endocytosis
• They are then re-secreted with a load of
cholesterol
• It transports this back to the liver
66
Receptor Mediated Endocytosis
• LDL – apoB-100, recognised by LDL receptors
• LDL receptors are present on cells which need to
take up cholesterol
• LDL binding to its receptor initiates endocytosis
• This brings the LDL and its receptor iside the cell,
within an endosome
• This endosome fuses with a lysosome
• The lysosome contains enzymes which hydrolyse
the cholesteryl esters to cholsterol and free fatty
acids
• apoB-100 is degraded to amino acids
67
Endocytosis of LDL
68
Receptor Mediated Endocytosis
• LDL also degraded to amino acids which
are released into cytosol
• LDL receptor is not degraded – it returns to
the cell surface
• apoB-100 present in VLDL, but it cannot
bind to the LDL receptor
• Conversion of VLDL to LDL exposes the
binding domain of apoB-100
69
70
ACAT
• Cholesterol which enters cell by receptor
mediated endocytosis can be re-esterified
by ACAT (acyl-CoA-cholesterol acyltransferase)
• Stored within cytosolic lipid droplets
71
Regulation of Blood Cholesterol
• Accumulation of excess cholesterol prevented by
reducing rate of cholesterol synthesis when
sufficient cholesterol is available from blood LDL
• Negative feedback inhibition
• High intracellular cholesterol levels activate ACAT,
increases esterification of cholesterol for storage
• High cellular cholesterol reduces transcription of the
gene which encodes LDL receptor
• Reduces production of receptor and thus uptake of
cholesterol from blood
72
73
Unregulated Cholesterol Production
• If more cholesterol is consumed in diet
and synthesised than is needed for
synthesis of membranes, bile salts and
steroids:
– accumulation of cholesterol in blood vessels
– These are called atherosclerotic plaques
– Obstruct blood flow leading to atherosclerosis
74
Atherosclerosis
• Blocked vessels, occluded coronary
arteries – heart attacks
• Major cause of death in industrialised
societies
• Atherosclerosis linked to high blood
cholesterol
• Also to high levels of LDL-bound receptor
• Negative correlation between HDL and
arterial disease
75
Familial Hypercholesterolemia
•
•
•
•
•
Genetic disorder
High blood cholesterol
Develop atherosclerosis in childhood
Defective LDL receptor
Receptor mediated uptake of cholesterol by LDL
does not occur
• Cholesterol is not cleared from blood
• Accumulated forming atherosclerotic plaques
• Endogenous cholesterol synthesis continues
despite excessive cholesterol in blood because
extracellular cholesterol cannot enter cell to
regulate intracellular synthesis
76
Treating Familial
Hypercholesterolemia
• Lovastatin and Compactin – derived from fungi
• These, and other analogues resemble
mevalonate
• Competitive inhibitors of HMG-CoA reductase
• Inhibit cholesterol synthesis
• Lovastatin can lower blood cholesterol up to 30 %
• Can combine drug with an edible resin which
binds bile salts to prevent their re-absorption –
makes drugs more effective
77
78
HDL Deficiency
•
•
•
•
•
•
•
•
HDL levels low in familial HDL deficiency (FHA)
HDL almost undetectable in Tangier disease
Both result from a mutation in ABC protein
Cholesterol depleted HDL can’t take on cholesterol
from cells that lack this protein
The cholesterol depleted HDL is rapidly removed from
the blood and destroyed
Both diseases rare
Teach us about role of ABC1 protein
Low plasma HDL correlates with high incidence of
coronary heart disease - ABC1 useful target for drugs
79
to control HDL levels
Intermediates in Cholesterol
Biosynthesis
• Isopentenyl pyrophosphate has other roles
apart from in cholesterol synthesis
• Precursor for synthesis of many
biomolecules
• Vitamins A,E and K, natural rubber,
essential oils and many others
• These compounds are called isoprenoids
80
Isoprenoid
Biosynthesis
81
Practice Questions
1. Which of the following is not an intermediate in the
synthesis of lanosterol from acetyl-CoA?
a)
b)
c)
d)
e)
Isopentenyl pyrophosphate
Malonyl-CoA
Mevalonate
Squalene
HMG-CoA
2. Cholesterol is synthesised from:
a)
b)
c)
d)
e)
acetyl-CoA
choline
lipoic acid
malate
oxalate
82
Practice Questions
3. A 30-carbon precursor of the steroid nucleus is:
a)
b)
c)
d)
e)
farnesyl pyrophosphate.
geranyl pyrophosphate.
isopentenyl pyrophosphate.
lysolecithin.
squalene.
4. Which of the following is derived from a sterol?
a)
b)
c)
d)
e)
Bile salts
Gangliosides
Geraniol
Phosphatidylglycerol
Prostaglandins
83
Practice Questions
5. Which of these statements about cholesterol
synthesis is true?
a) Cholesterol is the only known natural product whose
biosynthesis involves isoprene units.
b) Only half of the carbon atoms of cholesterol are derived
from acetate.
c) Squalene synthesis from farnesyl pyrophosphate results
in the release of two moles of PPi for each mole of
squalene formed.
d) The activated intermediates in the pathway are CDPderivatives.
e) The condensation of two five-carbon units to yield
geranyl pyrophosphate occurs in a “head-to-head”
fashion.
84
Practice Questions
6. Which of these statements about the regulation of
cholesterol synthesis is not true?
a) Cholesterol acquired in the diet has essentially no effect
on the synthesis of cholesterol in the liver.
b) Failure to regulate cholesterol synthesis predisposes
humans to atherosclerosis.
c) High intracellular cholesterol stimulates formation of
cholesterol esters.
d) Insulin stimulates HMG-CoA reductase.
e) Some metabolite or derivative of cholesterol inhibits
HMG-CoA reductase.
85
Practice Questions
7. What are the functions of cholesterol in humans?
8. Which is the commited step in cholesterol synthesis?
What enzyme catalyses this step?
9. How are the 2 activated isoprenes structurally related?
10. Which are the most dense lipoproteins? The least
dense? How does this relate to their lipid:protein ratio?
11. What part of the LDL particle is recognised by the LDL
receptor?
12. Based on the function of HDLs, why do you expect a
negative correlation between HDL levels and arterial
disease?
86