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Lipid Metabolism 3:
Cholesterol biosynthesis, lipoprotein
metabolism, steroid and eicosanoid synthesis
Bioc 460 Spring 2008 - Lecture 37 (Miesfeld)
Mike Brown and Joe
Goldstein shared the 1985
Nobel Prize for their work on
cholesterol metabolism
Cardiovascular disease is
caused by fatty acid deposits
that sequester cholesterol in
plaques that can break off
Steroids are cholesterol
derivatives that have been
used as anabolic enhancers
Key Concepts in Lipid Metabolism
•
Cholesterol is major component of membranes that is synthesized in liver cells
from acetyl-CoA through a complex reaction pathway involving six C5
intermediate isopentenyl pyrophosphate that form a C30 squalene product.
•
Cholesterol metabolism is an important component of cardiovascular disease
because of its association with the formation of atherosclerotic plaques. Over
70% of serum cholesterol is synthesized in the liver which is why statin drugs
target the cholesterol biosynthetic pathway.
•
Steroid hormones are cholesterol derivatives that bind to nuclear receptor
proteins in cells that function as gene-specific transcription factors. Steroid
hormones are important physiological ligands and pharmaceutical drugs.
•
Eicosanoids are fatty acid derivatives that are precursors to paracrine signaling
molecules such as prostaglandins, thromboxanes, and leukotrienes, all of which
are drug targets for various inflammatory diseases.
Cholesterol Biosynthesis
Cholesterol is a large hydrophobic molecule that controls membrane
fluidity in eukaryotes and is a metabolic precursor to steroid hormones.
The majority of serum-derived cholesterol comes from de novo
biosynthesis in the liver, not from dietary cholesterol. Therefore,
understanding cholesterol biosynthesis in relation to cardiovascular
disease has been a very active research area.
Radioisotope labeling experiments have shown that all 27 carbon atoms
of cholesterol are derived from acetyl-CoA.
Cholesterol Biosynthesis
involves the mevalonate pathway
and the isoprenoid pathway.
A key intermediate in the mevalonate
pathway is HMG-CoA
Cholesterol biosynthesis takes place in the
cytosol and consists of four distinct stages
In the first stage, three molecules of acetyl-CoA are used to make
mevalonate, a C6 compound that is the product of a reaction catalyzed by the
highly-regulated enzyme HMG CoA reductase.
Mevalonate is then phosphorylated and decarboxylated in stage 2 to form the
activated C5 isoprenoid intermediate, isopentenyl pyrophosphate. This
important metabolite is an intermediate in a number of other biosynthetic
reactions including those of plant chlorophylls and plant hormones.
In stage 3, three molecules of isopentenyl pyrophosphate are combined to
form farnesyl pyrophosphate (C15) which is then used to generate
squalene, a C30 cholesterol precursor.
In the fourth and final stage, squalene cyclicizes to form a four-ringed
molecule which is then modified by a number of reactions, ultimately
resulting in the loss of three methyl groups to generate cholesterol (C27), a
four-ringed sterol molecule.
Stage 1:
Generation of mevalonate
from acetyl-CoA
In the first reaction of this cytosolic
pathway, two molecules of acetyl-CoA
are condensed to form acetoacetyl-CoA
through a reaction catalyzed by the
enzyme thiolase. A third acetyl-CoA is
used to generate the C6 compound Hydroxy--methylglutaryl-CoA (HMGCoA) by the action of HMG-CoA
synthase. In what turns out to be the
rate-limiting step in the cholesterol
biosynthetic pathway, the enzyme HMGCoA reductase converts HMG-CoA to
mevalonate in a reduction reaction that
uses two molecules of NADPH and
releases coenzyme A.
Stage 2: Conversion of mevalonate to isopentenyl
pyrophosphate and dimethylallyl pyrophosphate
Mevalonate is activated by the addition of two phosphates groups donated
from ATP to generate 5-pyrophosphomevalonate.
The enzyme pyrophosphomevalonate decarboxylase then catalyzes
an ATP-dependent reaction that removes the terminal carboxyl group to
generate the C5 isoprenoid compound isopentenyl pyrophosphate
which is readily isomerized to form dimethylallyl pyrophosphate.
isopentenyl pyrophosphate
dimethylallyl pyrophosphate
Stage 2: Conversion of mevalonate to isopentenyl
pyrophosphate and dimethylallyl pyrophosphate
1 C6
1 C5
isomerize
or
1 C5
Stages 3 and 4: formation of cholesterol (C27)
from six C5 isoprenoids
Prenyl transferase catalyzes a condensation reaction in which isopentenyl
pyrophosphate and dimethylallyl pyrophosphate condense in a head to tail fashion
to form the C10 compound geranyl pyrophosphate. The same enzyme adds a
second isopentenyl pyrophosphate to generate farnesyl pyrophosphate.
Two molecules of farnesyl pyrophosphate are then linked in a head to head
arrangement by the enzyme squalene synthase to form squalene (C30) in a
reduction reaction using NADPH.
The conversion of a C30 hydrocarbon chain into a cyclic cholesterol molecule
involves a complex set of reactions that are catalyzed by enzymes called
cyclases that promote the formation of the four-ringed cholesterol precursor
lanosterol.
In the final steps of the pathway, lanosterol is converted to cholesterol by a series
of 19 reactions that involve additional bond rearrangements, and ultimately, the
removal of three carbons to generate the C27 cholesterol product.
1 C5
Stages 3 and 4: formation of cholesterol (C27)
from six C5 isoprenoids
+
1 C5
1 C30
1 C10
+1 C5
1 C15
+1 C15
1 C27
1 C30
Cholesterol synthesized
in the liver has three
metabolic fates; stored,
exported, or converted to
bile acid
Cholesterol can be esterified with a fatty
acid by the enzyme acyl-CoAcholesterol acyl transferase (ACAT) to
make cholesterol esters that are stored in
lipid droplets, or packaged into lipoprotein
particles and exported to the peripheral
tissues. As much as 50% of the
cholesterol synthesized in liver cells on a
daily basis is converted to bile acids
which are transported to the bile duct and
secreted into the small intestine to aid in
fat absorption.
Cholesterol synthesized
in the liver has three
metabolic fates; stored,
exported, or converted to
bile acid
What would happen to
cholesterol levels in the liver if
bile acids were excreted rather
than recycled back to the liver?
Bile acid resin
Metabolism of dietary fats and cholesterol
Triacylglycerols and cholesterol are transported through the circulatory
system as components of plasma lipoprotein particles that consist of
membrane-bound vesicles containing a hydrophobic core and one or
more proteins on the surface called apolipoproteins.
Metabolism of dietary fats and cholesterol
Chylomicrons contain mostly triacylglycerols in their hydrophobic core and a
low amount of protein relative to lipid, thereby giving them the lowest density. In
contrast, high density lipoproteins (HDL), have a high percentage of protein
compared to lipid and are very small resulting in high density. The three other
lipoprotein classes, very low density lipoproteins (VLDL), intermediate density
lipoproteins (IDL), also called VLDL remnants, and low density lipoproteins
(LDL), are progressively smaller and contain higher ratios of protein to lipid.
Metabolism of dietary fats and cholesterol
Lipoproteins function in the body to transport lipids (triacylglycerols and
cholesterol) from the small intestine or the liver out to peripheral tissues
and then back again to the liver. The level of lipoproteins in blood plasma
is significant after a lipid-rich meal as evidenced by the milky appearance
of blood due to the high numbers of chylomicrons. Cholesterol deposits in
blood vessels are considered one of the major culprits in cardiovascular
disease, and therefore, control of cholesterol homeostasis is an
important regulatory process.
Metabolism of dietary fats and cholesterol
Steady state levels of serum cholesterol are determined by cholesterol recycling
via lipoprotein particles, and by cholesterol excretion. Lipoproteins, primarily
VLDL particles, transport the triacylglycerol and cholesterol synthesized in liver
cells (or deposited there by the chylomicron remnants) out to the peripheral
tissues.
Like chylomicrons, VLDL particles contain apoC-II and ApoE on the surface to
facilitate triacylglycerol delivery to the tissues, but they also contain apolipoprotein
B-100, an apolipoprotein that is recognized by another cell surface receptor called
the LDL receptor.
About half of the VLDL particles are converted to IDL particles (VLDL remnants)
that are returned to the liver, and the rest continue to circulate until nearly all of
the triacylglycerol is removed and they are converted to LDL particles.
These cholesterol-rich lipoproteins are called "bad cholesterol" and are
Metabolism of dietary fats and cholesterol
"Why is LDL considered "bad cholesterol" and
HDL "good cholesterol?"
The answer is that high
LDL levels in the serum
are clinically associated
with an increased risk of
atherosclerosis,
whereas, high HDL
levels in the serum are
associated with a
significantly decreased
risk of atherosclerosis.
Therefore it is "bad" to
have too much LDL in
the serum and "good" to
have elevated levels of
HDL.
"Why is LDL considered "bad cholesterol" and
HDL "good cholesterol?"
Cardiovascular disease is due to the build-up of
cholesterol-rich fibrous plaques in arteries
Atherosclerotic plaques are the result of infiltration of LDL particles into the space
below the endothelial lining of blood vessels. This process initiates an immune
response that triggers the local accumulation of inflammatory cells that
degrade LDL particles causing the cholesterol to be released and taken up
by a type of macrophage cell called a foam cell. The site of inflammation grows
as more LDL particles attach to the plaque, eventually resulting in smooth
muscle cell proliferation and calcification. Thrombosis can also block arteries.
LDL receptor cycling and cholesterol homeostasis
The connection between high serum LDL levels and atherosclerosis was
made by Brown and Goldstein when they discovered that a genetic
disorder called familial hypercholesterolemia (FH) was due to
mutations in the LDL receptor gene. The cholesterol esters are
released from lysosomes and stored in cholesterol droplets or used to
synthesize bile acids or steroids.
The intracellular pool of cholesterol is determined by both cholesterol
ester uptake from LDL particles and de novo cholesterol
biosynthesis. Low cholesterol levels stimulate HMG-CoA reductase
activity and induce expression of the LDL receptor gene through
protealytic activation of sterol regulatory element binding proteins
(SREBPs) that are transcription factors that regulate the expression of
numerous lipid metabolizing genes.
Statin drugs inhibit cholesterol biosynthesis by
blocking the activity of HMG-CoA reductase
Since de novo synthesis of liver cholesterol accounts for the majority of
cholesterol in our bodies (~70% for most individuals), pharmaceutical companies
first developed drugs that could be used to inhibit cholesterol synthesis and
thereby activate LDL receptor expression. The result of this treatment strategy is
decreased serum LDL levels due to uptake by liver cells; decreased serum LDL
levels is associated with reduced risk of cardiovascular disease.
4
How do statin
drugs lower
serum LDL
levels?
Why would this
decrease the risk
of cardiovascular
disease?
1
3
2
Ezetimibe (Zetia) blocks dietary cholesterol uptake
Zetia was found to block a
cholesterol transporter in the
small intestine called NiemannPick C1 Like 1 (NPC1L1) protein.
By disrupting cholesterol
absorption in the intestine, it is
possible to block the cycle of
cholesterol export and import to
the liver through formation of bile
acids. The result is that
cholesterol is excreted causing
more cholesterol in the liver to be
shunted toward bile acid
synthesis. Ezetimibe can be used
in combination with statins to
achieve an additive effect on
serum LDL and HDL levels.
Ezetimibe (Zetia) blocks dietary cholesterol uptake
A cholesterol-lowering drug of this type is
Vytorin which contains both ezetimibe
and simvastatin and is capable of
lowering serum LDL levels by 50% and
increasing HDL levels by as much as
10%. Vytorin contains ezetimibe and
simvastatin.
Surprisingly, recent clinical studies
have shown that Vytorin, a Merck
collaborative drug, is no more effective
in reducing cardiovascular disease
than statin drugs alone, eventhough
Vytorin lowers serum cholesterol levels
much more than either ezetimibe or
simvastatin. It is not clear what these
new results mean except that serum
cholesterol levels alone may not be the
best predictor of cardiovascular disease.
Steroid hormones are synthesized from cholesterol
The five major hormones in mammals
are all derived from modifications of
cholesterol following removal of the
side chain at the C-17 carbon of the D
ring to produce pregnenolone. This
C21 sterol gives rise to progesterone
which can be converted to cortisol,
androstenedione and corticosterone.
Cortisol is a glucocorticoid hormone
that regulates liver metabolism.
Androstenedione is an androgenic
hormone that gives rise to testosterone
which is then converted to either
estradiol or dihydrotesterone.
Aldosterone, a mineralocorticoid that
regulates ion transport in the kidney, is
derived from corticosterone.
Steroid hormones are synthesized from cholesterol
The three other major sites of steroid synthesis are the ovaries in females
(estrogen), testes in males (testosterone), and the corpus luteum in pregnant
females (progesterone). Importantly, the adrenal glands also synthesizes
androgens, which is how females acquire testosterone for estradiol production.
The chemical and molecular structure of steroid hormones, as well as, the
structure of the steroid hormone binding domain of a steroid receptor protein, are
shown below. Note the similar structure to cholesterol and the hydrophobic
nature of these important signaling molecules.
Steroid hormones are synthesized from cholesterol
Subtle changes in the side
groups of steroid hormones
account for differences in their
agonist (activating) or
antagonist (inhibiting) functions
as pharmacological agents.
Dihydrotestosterone is a
naturally occurring androgen,
whereas, nandrolone is a
potent synthetic agonist that
has been abused by
bodybuilders to gain muscle
mass. Finasteride, a
testosterone antagonist used to
treat male pattern baldness, a
condition caused by high levels
of testosterone in the hair
follicle cells.
Eicosanoids
Biosynthesis
Eicosanoids are a group
of signaling molecules
derived from C20
polyunsaturated fatty
acids such as
arachidonate that are
released from the
membrane by
phospholipases. The
three major classes of
arachidonate-derived
eicosanoids are
prostaglandins,
thromboxanes and
leukotrienes.
COX-2 inhibitors
COX-1 is involved in producing
prostaglandins that stimulate
mucin secretion and protect the
lining of the stomach from high
pH, COX-2 expression is
specifically induced by
inflammatory signals. Aspirin,
ibuprofen, and naproxen, are all
non-selective NSAIDs that
inhibit both COX-1 and COX-2.
Vioxx and Celebrex selectively
inhibit COX-2, but they also
partially inhibit prostacyclin
synthesis which can lead to clot
formation. Vioxx is no longer
available because of an
increased risk of heart attacks.