Introduction to Cholesterol Metabolism

Biosynthesis of Cholesterol
Regulation of Cholesterol Synthesis
Proteolytic Regulation of HMG-CoA Reductase
Utilization of Cholesterol
Cytochrome P450 Enzymes in Cholesterol Metabolism
Regulation of Cellular Sterol Content: SREBP
Serum Cholesterol Values
Treatment of Hypercholesterolemia

Bile Acid Synthesis and Metabolism

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Introduction to Cholesterol Metabolism

Cholesterol is an extremely important biological molecule that has roles in
membrane structure as well as being a precursor for the synthesis of the steroid
hormones, the bile acids, and vitamin D. Both dietary cholesterol, and that
synthesized de novo, are transported through the circulation in lipoprotein particles.
The same is true of cholesteryl esters, the form in which cholesterol is stored in
cells. Due to its important role in membrane function, all cells express the enzymes
of cholesterol biosynthesis.
 The synthesis and utilization of cholesterol must be tightly regulated in order to
prevent over-accumulation and abnormal deposition within the body. Of particular
clinical importance is the abnormal deposition of cholesterol and cholesterol-rich
lipoproteins in the coronary arteries. Such deposition, eventually leading to
atherosclerosis, is the leading contributory factor in diseases of the coronary
arteries.

Cholesterol

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Biosynthesis of Cholesterol
 Slightly less than half of the cholesterol in the body derives from biosynthesis
de novo. Biosynthesis in the liver accounts for approximately 10%, and in the
intestines approximately 15%, of the amount produced each day. The cholesterol
biosynthesis pathway involves enzymes that are in the cytoplasm, microsomes
(ER), and peroxisomes. Synthesis of cholesterol, like that of most biological lipids,
begins from the two-carbon acetate group of acetyl-CoA.
The acetyl-CoA utilized for cholesterol biosynthesis is derived from an oxidation
reaction (e.g., fatty acids or pyruvate) in the mitochondria and is transported to the
cytoplasm by the same process as that described for fatty acid synthesis (see the
Figure below). Acetyl-CoA can also be synthesized from cytosolic acetate derived
from cytoplasmic oxidation of ethanol which is initiated by cytoplasmic alcohol
dehydrogenase (ADH). All the reduction reactions of cholesterol biosynthesis use
NADPH as a cofactor. The isoprenoid intermediates of cholesterol biosynthesis can
be diverted to other synthesis reactions, such as those for dolichol (used in the
synthesis of N-linked glycoproteins, coenzyme Q (of the oxidative phosphorylation
pathway) or the side chain of heme-a. Additionally, these intermediates are used in
the lipid modification of some proteins.
 Pathway for the movement of acetyl-CoA units from within the
mitochondrion to the cytoplasm. Note that the cytoplasmic malic enzyme
catalyzed reaction generates NADPH which can be used for reductive biosynthetic
reactions such as those of fatty acid and cholesterol synthesis. SLC25A1 is the
citrate transporter (also called the dicarboxylic acid transporter). Transport of
pyruvate across the plasma membrane is catalyzed by the SLC16A1 protein (also
called the monocarboxylic acid transporter 1, MCT1) and transport across the outer
mitochondrial membrane involves a voltage-dependent porin transporter. Pyruvate
transport across the inner mitochondrial membrane requires a heterotetrameric
transport complex (mitochondrial pyruvate carrier) consisting of the MPC1 gene and
MPC2 gene encoded proteins.

The process of cholesterol synthesis can be considered to be composed of five

major steps:
1. Acetyl-CoAs are converted to 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA)
2. HMG-CoA is converted to mevalonate
3. Mevalonate is converted to the isoprene based molecule, isopentenyl
pyrophosphate (IPP), with the concomitant loss of CO2
4. IPP is converted to squalene
5. Squalene is converted to cholesterol.
 Pathway of cholesterol biosynthesis. Synthesis of cholesterol begins with the
transport of acetyl-CoA from within the mitochondria to the cytosol. The rate limiting
step in cholesterol synthesis occurs at the 3-hydroxy-3-methylglutaryl-CoA (HMG-
CoA) reducatase, HMGR, catalyzed step. The phosphorylation reactions are
required to solubilize the isoprenoid intermediates in the pathway. Intermediates in
the pathway are used for the synthesis of prenylated proteins, dolichol, coenzyme Q
and the side chain of heme a. The abbreviation "PP" (e.g. isopentenyl-PP) stands
for pyrophosphate. ACAT2: acetyl-CoA acetyltransferase 2. HMGCS1: HMG-CoA
synthase 1 (cytosolic). HMGCR: HMG-CoA reductase. MVK: mevalonate kinase.
PMVK: phosphomevalonate kinase. MVD: diphosphomevalonate decarboxylase.
IDI1/IDI2: isopentenyl-diphosphate delta isomerase 1 and 2. FDPS: farnesyl
diphosphate synthase. GGPS1: geranylgeranyl diphosphate synthase 1. FDFT1:
farnesyl-diphosphate farnesyltransferase 1 (more commonly called squalene
synthase). SQLE: squalene epoxidase (also called squalene monooxygenase). LSS:
lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase). DHCR7: 7-
dehydrocholesterol reductase.

HMG-CoA Synthesis
Acetyl-CoA units are converted to mevalonate by a series of reactions that
begins with the formation of HMG-CoA. Unlike the HMG-CoA formed during ketone
body synthesis in the mitochondria, this form is synthesized in the cytoplasm.
However, the pathway and the necessary enzymes are similar to those in the
mitochondria. Two moles of acetyl-CoA are condensed in a reversal of the thiolase
reaction, forming acetoacetyl-CoA. The cytoplasmic thiolase enzyme involved in
cholesterol biosynthesis is acetoacetyl-CoA thiolase (acetyl-CoA acetyltransferase
2) encoded by the ACAT2 gene. Although the bulk of acetoacetyl-CoA is derived via
this process, it is possible for some acetoacetate, generated during ketogenesis, to
diffuse out of the mitochondria and be converted to acetoacetyl-CoA in the cytosol
via the action of acetoacetyl-CoA synthetase (AACS). Acetoacetyl-CoA and a third
mole of acetyl-CoA are converted to HMG-CoA by the action of the cytosolic version
of HMG-CoA synthase encoded by the HMGCS1 gene. The HMGCS1 gene is
located on chromosome 5p12 and is composed of 12 exons that generate two
alternatively spliced mRNAs that encode two different isoforms: isoform 1 (520
amino acids) and isoform 2 (478 amino acids).
Mevalonate Synthesis
HMG-CoA is then converted to mevalonate by HMG-CoA reductase, HMGR
(this enzyme is bound in the endoplasmic reticulum, ER). HMGR absolutely requires
NADPH as a cofactor and two moles of NADPH are consumed during the
conversion of HMG-CoA to mevalonate. The reaction catalyzed by HMGR is the rate
limiting step of cholesterol biosynthesis, and this enzyme is subject to complex
regulatory controls as discussed below. HMGR is derived from the HMGCR gene
which is located on chromosome 5q13.3 and is composed of 22 exons that generate
two alternatively spliced mRNAs that encode HMGR isoform 1 (888 amino acids)
and HMGR isoform 2 (835 amino acids).
 Mevalonate is then activated by two successive phosphorylations (catalyzed by
mevalonate kinase, and phosphomevalonate kinase) yielding, sequentially,
mevalonate 5-phosphate and then mevalonate 5-diphosphate (the latter compound
is also called 5-pyrophosphomevalonate or mevalonate 5-pyrophosphate). In
humans, mevalonate kinase is a peroxisome localized enzyme encoded by the MVK
gene. The MVK gene is located on chromosome 12q24 and is composed of 12
exons that generate three alternatively spliced mRNAs. Phosphomevalonate kinase
is also a peroxisomal enzyme and it is derived from the PMVK gene. The PMVK
gene is located on chromosome 1q22 and is composed of 6 exons that encode a
192 amino acid protein.
Isopentenylpyrophosphate (IPP) Synthesis
Following the formation of mevalonate 5-diphosphate, an ATP-dependent
decarboxylation yields isopentenylpyrophosphate (IPP) which is an activated
isoprenoid molecule. The synthesis of IPP is catalyzed by diphosphomevalonate
decarboxylase (also called mevalonate-5-pyrophosphate decarboxylase) derived
from the MVD gene. The MVD gene is located on chromosome 16q24.3 and is
composed of 13 exons that encode a 400 amino acid protein. Isopentenyl
pyrophosphate is in equilibrium with its isomer, dimethylallyl pyrophosphate (DMPP)
via the action of isopentenyl-diphosphate delta isomerase (also called
isopentenylpyrophosphate isomerase). Humans express two isopentenyl-
diphosphate delta isomerase genes, IDI1 and IDI2. The IDI1 gene is located on
chromosome 10p15.3 and is composed of 7 exons that generate four alternatively
spliced mRNAs that collectively encode three protein isoforms that are localized to
the peroxisomes. The IDI2 gene is located on the same chromosomal region as the
IDI1 gene but is composed of only 5 exons and encodes a 227 amino acid protein.
Squalene Synthesis
One molecule of IPP condenses with one molecule of DMPP to generate
geranyl pyrophosphate, GPP. GPP further condenses with another IPP molecule to
yield farnesyl pyrophosphate, FPP. Synthesis of both GPP and FPP is catalyzed by
the enzyme, farnesyl diphosphate synthase. Farnesyl diphosphate synthase is
derived from the FDPS gene which is located on chromosome1q22 and is
composed of 11 exons that generate five alternatively spliced mRNAs that, together,
encode three different isoforms of the enzyme.
The synthesis of squalene, from FPP, represents the first cholesterol-specific
step in the cholesterol synthesis pathway. This is due to the fact that, as depicted in
the pathway Figure above, several intermediates in the pathway can be diverted to
the production of other biologically relevant molecules. The synthesis of squalene is
catalyzed by the NADPH-requiring enzyme, farnesyl-diphosphate
farnesyltransferase 1 (commonly called squalene synthase). Farnesyl-diphosphate
farnesyltransferase 1 (encoded by the FDFT1 gene) catalyzes the two-step head-to-
head condensation of two molecules of FPP, yielding squalene. The FDFT1 gene is
located on chromosome 8p23.1 and is composed of 14 exons that generate 11
alternatively spliced mRNAs. These 11 different FDFT1-encoded mRNAs
 collectively synthesize five different isoforms of farnesyl-diphosphate
farnesyltransferase 1.
Squalene to Cholesterol
Squalene then undergoes a two step cyclization to yield lanosterol. This first
reaction in this two-step cyclization is catalyzed by the enzyme, squalene epoxidase
(also called squalene monooxygenase). This enzyme uses NADPH as a cofactor to
introduce molecular oxygen as an epoxide at the 2,3 position of squalene forming
the intermediate, 2,3-oxidosqualene. In the second step, this epoxide intermediate is
converted to lanosterol through the action of the enzyme lanosterol synthase (2,3-
oxidosqualene-lanosterol cyclase). Squalene epoxidase is derived from the SQLE
gene which is located on chromosome 8q24.13 and is composed of 12 exons that
encode a protein of 574 amino acids. Lanosterol synthase is derived from the LSS
gene which is located on chromosome 21q22.3 and is composed of 25 exons that
generate four alternatively spliced mRNAs which together generate three distinct
isoforms of the enzyme.
Through a series of 19 additional reactions, lanosterol is converted to
cholesterol. These 19 reaction steps are catalyzed by nine different enzymes that
are localized either to the ER or to the peroxisomes. The terminal reaction in
cholesterol biosynthesis is catalyzed by the enzyme 7-dehydrocholesterol reductase
encoded by the DHCR7 gene. Functional DHCR7 protein is a 55.5 kDa NADPH-
requiring integral membrane protein localized to the microsomal (ER) membrane.
The DHCR7 gene is located on chromosome 11q13.4 and is composed of 9 exons
that generate two alternatively spliced mRNAs, both of which encode the same 475
amino acid protein. Deficiency in DHCR7 (due to gene mutations) results in the
disorder called Smith-Lemli-Opitz syndrome, SLOS. SLOS is characterized by
increased levels of 7-dehydrocholesterol and reduced levels (15% to 27% of normal)
of cholesterol resulting in multiple developmental malformations and behavioral
problems.
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Regulating Cholesterol Synthesis

Normal healthy adults synthesize cholesterol at a rate of approximately 1g/day
and consume approximately 0.3g/day. A relatively constant level of cholesterol in
the blood (150–200 mg/dL) is maintained primarily by controlling the level of de
novo synthesis. The level of cholesterol synthesis is regulated in part by the dietary
intake of cholesterol. Cholesterol from both diet and synthesis is utilized in the
formation of membranes and in the synthesis of the steroid hormones and bile
acids. The greatest proportion of cholesterol is used in bile acid synthesis.
The cellular supply of cholesterol is maintained at a steady level by three
distinct mechanisms:
1. Regulation of HMGR activity and levels
 2. Regulation of excess intracellular free cholesterol through the activity of
sterol O-acyltransferases, SOAT1 and SOAT2 with SOAT2 being the
predominant activity in liver. The original designation for these enzymes was
ACAT for acyl-CoA: cholesterol acyltranferase. However, this conflicts with the
official ACAT enzymes, ACAT1 and ACAT2 which are acetyl-CoA
acetyltransferases 1 and 2. These latter two enzymes are thiolases discussed
in the Lipolysis and Fatty Acid Oxidation page.
3. Regulation of plasma cholesterol levels via LDL receptor-mediated uptake
and HDL-mediated reverse transport.
Regulation of HMGR activity is the primary means for controlling the level of
cholesterol biosynthesis. The enzyme is controlled by four distinct mechanisms:
feed-back inhibition, control of gene expression, rate of enzyme degradation and
phosphorylation-dephosphorylation.
The first three control mechanisms are exerted by cholesterol itself. Cholesterol
acts as a feed-back inhibitor of pre-existing HMGR as well as inducing rapid
degradation of the enzyme. The latter is the result of cholesterol-induced
polyubiquitination of HMGR and its degradation in the proteosome (see proteolytic
degradation below). This ability of cholesterol is a consequence of the sterol
sensing domain, SSD of HMGR. In addition, when cholesterol is in excess the
amount of mRNA for HMGR is reduced as a result of decreased expression of the
gene. The mechanism by which cholesterol (and other sterols) affect the
transcription of the HMGR gene is described below under regulation of sterol
content.
Regulation of HMGR through covalent modification occurs as a result of
phosphorylation and dephosphorylation. The enzyme is most active in its unmodified
form. Phosphorylation of the enzyme decreases its activity. HMGR is
phosphorylated by AMP-activated protein kinase, AMPK (this is not the same as
cAMP-dependent protein kinase, PKA). AMPK itself is activated via phosphorylation.
Phosphorylation of AMPK is catalyzed by at least two enzymes. The primary kinase
responsible activation of AMPK is LKB1 (liver kinase B1). LKB1 was first identified
as a gene in humans carrying an autosomal dominant mutation in Peutz-Jeghers
syndrome, PJS. LKB1 is also found mutated in lung adenocarcinomas. The second
AMPK phosphorylating enzyme is calmodulin-dependent protein kinase kinase-beta
(CaMKKβ). CaMKKβ induces phosphorylation of AMPK in response to increases in
intracellular Ca2+ as a result of muscle contraction. Visit AMPK: The Master
Metabolic Regulator for more detailed information on the role of AMPK in regulating
metabolism.
 Regulation of HMGR by covalent modification. HMGR is most active in the
dephosphorylated state. Phosphorylation (Ser872) is catalyzed by AMP-activated
protein kinase (AMPK) an enzyme whose activity is also regulated by
phosphorylation. Phosphorylation of AMPK is catalyzed by at least two enzymes:
LKB1 and CaMKKβ. Hormones such as glucagon and epinephrine negatively affect
cholesterol biosynthesis by increasing the activity of the specific regulatory subunits
of the protein phosphatase 1 (PP1) family enzymes. Humans express three PP1
catalytic subunit genes (PPP1CA, PPP1CB, and PPP1CC) and 181 different
regulatory or PP1-interacting proteins. The original designation for a regulatory
subunit of PP1 was called phosphoprotein phosphatase inhibitor-1, PPI-1. Opposing
the effects of glucagon and epinephrine, insulin stimulates the removal of
 phosphates and, thereby, activates HMGR activity. Additional regulation of HMGR
occurs through cholesterol-mediated feedback inhibition, as well as regulation of its
synthesis by elevation in intracellular cholesterol and sterol levels. This latter
phenomenon involves the transcription factor SREBP described below.

The activity of HMGR is additionally controlled by the cAMP signaling pathway.

Increases in cAMP lead to activation of cAMP-dependent protein kinase, PKA. In the
context of HMGR regulation, PKA phosphorylates a regulatory subunit of
phosphoprotein phosphatase 1 (PP1) leading to an increase in its PP1 inhibitory
activity. The large family of protein phosphatase regulatory subunits regulate and/or
inhibit the activity of numerous phosphatases including members of the protein
phosphatase 2A (PP2A) family which remove phosphates from AMPK and HMGR.
Phosphatases of the protein phosphatase 2C (PP2C) family also remove
phosphates from AMPK. This maintains AMPK in the phosphorylated and active
state, and HMGR in the phosphorylated and inactive state. As the stimulus leading
to increased cAMP production is removed, the level of phosphorylations decreases
and that of dephosphorylations increases. The net result is a return to a higher level
of HMGR activity.
Since the intracellular level of cAMP is regulated by hormonal stimuli, regulation
of cholesterol biosynthesis is hormonally controlled. Insulin leads to a decrease in
cAMP, which in turn activates cholesterol synthesis. Alternatively, glucagon and
epinephrine, which increase the level of cAMP, inhibit cholesterol synthesis.
The ability of insulin to stimulate, and glucagon to inhibit, HMGR activity is
consistent with the effects of these hormones on other metabolic pathways. The
basic function of these two hormones is to control the availability and delivery of
energy to all cells of the body.
Long-term control of HMGR activity is exerted primarily through control over the
synthesis and degradation of the enzyme. When levels of cholesterol are high, the
level of expression of the HMGR gene is reduced. Conversely, reduced levels of
cholesterol activate expression of the gene. Insulin also brings about long-term
regulation of cholesterol metabolism by increasing the level of HMGR synthesis.
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Proteolytic Regulation of HMG-CoA Reductase

The stability of HMGR is regulated as the rate of flux through the mevalonate
synthesis pathway changes. When the flux is high the rate of HMGR degradation is
also high. When the flux is low, degradation of HMGR decreases. This phenomenon
can easily be observed in the presence of the statin drugs as discussed below.
HMGR is localized to the ER and like SREBP (see below) contains a sterol-
sensing domain, SSD. When sterol levels increase in cells there is a concomitant
increase in the rate of HMGR degradation. The degradation of HMGR occurs within
the proteosome, a multiprotein complex dedicated to protein degradation. The
 primary signal directing proteins to the proteosome is ubiquitination. Ubiquitin is a
7.6kDa protein that is covalently attached to proteins targeted for degradation by
ubiquitin ligases. These enzymes attach multiple copies of ubiquitin allowing for
recognition by the proteosome. HMGR has been shown to be ubiquitinated prior to
its degradation. The primary sterol regulating HMGR degradation is cholesterol
itself. As the levels of free cholesterol increase in cells, the rate of HMGR
degradation increases.
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The Utilization of Cholesterol

Cholesterol is transported in the plasma predominantly as cholesteryl esters
associated with lipoproteins. Dietary cholesterol is transported from the small
intestine to the liver within chylomicrons. Cholesterol synthesized by the liver, as
well as any dietary cholesterol in the liver that exceeds hepatic needs, is transported
in the serum within LDLs. The liver synthesizes VLDLs and these are converted to
LDLs through the action of endothelial cell-associated lipoprotein lipase. Cholesterol
found in plasma membranes can be extracted by HDLs and esterified by the HDL-
associated enzyme lecithin-cholesterol acyltransferase, LCAT. The cholesterol
acquired from peripheral tissues by HDLs can then be transferred to VLDLs and
LDLs via the action of cholesteryl ester transfer protein (CETP) which is associated
with HDLs. Reverse cholesterol transport allows peripheral cholesterol to be
returned to the liver in LDLs. Ultimately, cholesterol is excreted in the bile as free
cholesterol or as bile salts following conversion to bile acids in the liver.
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Cytochrome P450 Enzymes in Cholesterol Metabolism

Cytochrome P450 enzymes are involved in a diverse array of biological
processes that includes lipid, cholesterol, and steroid metabolism as well as the
metabolism of xenobiotics. The now common nomenclature used to designate P450
enzymes is CYP. There are at least 57 CYP enzymes in human tissues with eight
being involved in cholesterol biosynthesis and metabolism, which includes
conversion of cholesterol to bile acids. CYP metabolism of cholesterol yields several
oxysterols that function as biologically active molecules such as in the activation of
the liver X receptors (LXRs) and SREBP (see the next section).
CYP3A4: CYP3A4 is also known as glucocorticoid-inducible P450 and
nifedipine oxidase. Nifedipine is a member of the calcium channel blocker
drugs used to treat hypertension. CYP3A4 is a major hepatic P450 enzyme
and is responsible for the biotransformation of nearly 60% of all commercially
available drugs. With respect to cholesterol metabolism, CYP3A4 catabolizes
cholesterol to 4β-hydroxycholesterol. This cholesterol derivative is one of the
major circulating oxysterols and is seen at elevated levels in patients treated
with anti-seizure medications such as carbamazepine, phenobarbitol, and
phenytoin. The nuclear receptor, pregnane X receptor (PXR), is known to be
an inducer of the CYP3A4 gene.
CYP7A1: CYP7A1 is also known as cholesterol 7α-hydroxylase and is the
rate limiting enzyme in the primary pathway of bile acid synthesis referred to
as the classic pathway. This reaction of bile acid synthesis plays a major role
in hepatic regulation of overall cholesterol balance. Deficiency in CYP7A1
manifests with markedly elevated total cholesterol as well as LDL, premature
gallstones, premature coronary and peripheral vascular disease. Treatment of
this disorder with members of the statin drug family do not alleviated the
elevated serum cholesterol due to the defect in hepatic diversion of cholesterol
into bile acids.
CYP7B1: CYP7B1 is also known as oxysterol 7α-hydroxylase and is involved
in the synthesis of bile acids via the less active secondary pathway referred to
as the acidic pathway. A small percentage (1%) of individuals suffering from
autosomal recessive hereditary spastic paraplegia 5A (SPG5A) have been
shown to harbor mutations in the CYP7B1 gene.
CYP8B1: CYP8B1 is also known as sterol 12a-hydroxylase and is involved in
the conversion of 7-hydroxycholesterol (CYP7A1 product) to cholic acid which
is one of two primary bile acids and is derived from the classic pathway of bile
acid synthesis. The activity of CYP8B1 controls the ratio of cholic acid over
chenodeoxycholic acid in the bile.
CYP27A1: CYP27A1 is also known as sterol 27-hydroxylase and is localized
to the mitochondria. CYP27A1 functions with two cofactor proteins called
adrenodoxin and adrenodoxin reductase to hydroxylate a variety of sterols at
the 27 position. CYP27A1 is also involved in the diversion of cholesterol into
bile acids via the less active secondary pathway referred to as the acidic
pathway. Deficiencies in CYP27A1 result in progressive neurological
dysfunction, neonatal cholestasis, bilateral cataracts, and chronic diarrhea.
CYP39A1: CYP39A1 is also known as oxysterol 7α-hydroxylase 2. This P450
enzyme was originally identified in mice in which the CYP7B1 gene had been
knocked out. The preferential substrate for CYP39A1 is 24-
hydroxycholesterol, which is a major product of CYP46A1, which via
CYP39A1 action is diverted into bile acid synthesis.
CYP46A1: CYP46A1 is also known as cholesterol 24-hydroxylase. This
enzyme is expressed primarily in neurons of the central nervous system where
it plays an important role in metabolism of cholesterol in the brain. The product
of CYP46A1 action if 24S-hydroxycholesterol which can readily traverse the
blood-brain-barrier to enter the systemic circulation. This pathway of
cholesterol metabolism in the brain is a part of the reverse cholesterol
transport process and serves as a major route of cholesterol turnover in the
brain. 24S-hydroxycholesterol is a known potent activator of LXR and as such
 serves as an activator of the expression of LXR target genes and thus, can
effect regulation of overall cholesterol metabolism not only in the brain but
many other tissues as well.
CYP51A1: CYP51A1 is also referred to as lanosterol-14α-demethylase. This
P450 enzyme is the only one of the eight that is involved in de novo
cholesterol biosynthesis and it catalyzes the removal of the 14α-methyl group
from lanosterol resulting in the generation of at least two oxysterols that, in
mammalian tissues, are efficiently converted into cholesterol as well as more
polar sterols and steryl esters. The oxysterols derived through the action of
CYP51A1 inhibit HMGR and are also known to inhibit sterol synthesis. Knock-
out of the mouse CYP51A1 homolog results in a phenotype similar to that
seen in the human disorder known as Antley-Bixler syndrome (ABS). ABS
represents a group of heterogeneous disorders characterized by skeletal,
cardiac, and urogenital abnormalities that have frequently been associated
with mutations in the fibroblast growth factor receptor 2 (FGFR2) gene.
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Regulation of Cellular Sterol Content

The continual alteration of the intracellular sterol content occurs through the
regulation of key sterol synthetic enzymes as well as by altering the levels of cell-
surface LDL receptors. As cells need more sterol they will induce their synthesis and
uptake, conversely when the need declines synthesis and uptake are decreased.
Regulation of these events is brought about primarily by sterol-regulated
transcription of key rate limiting enzymes and by the regulated degradation of
HMGR. Activation of transcriptional control occurs through the regulated cleavage of
the membrane-bound transcription factor sterol regulated element binding protein,
SREBP. As discussed above, degradation of HMGR is controlled by the ubiquitin-
mediated pathway for proteolysis.
Sterol control of transcription affects more than 30 genes involved in the
biosynthesis of cholesterol, triacylglycerols, phospholipids and fatty acids.
Transcriptional control requires the presence of an octamer sequence in the gene
termed the sterol regulatory element, SRE-1. It has been shown that SREBP is the
transcription factor that binds to SRE-1 elements. Humans express two distinct
SREBP genes. These genes are identified as sterol regulatory element binding
transcription factor 1 (SREBF1) and sterol regulatory element binding transcription
factor 2 (SREBF2). In addition, mammalian SREBF1 encodes two major proteins
identified as SREBP-1a and SREBP-1c/ADD1 (ADD1 is adipocyte differentiation-1)
as a consequence of alternative transcriptional start sites resulting in the utilization
of different first exons that are spliced to a common exon 2. The SREBF1 gene is
located on chromosome 17p11.2 and is composed of 21 exons. The human
SREBP-1a protein (1147 amino acids) predominates in the spleen and intestines
while the SREBP-1c protein (1123 amino acids) predominates in liver, adipose
tissue, and muscle. The SREBF2 gene is located on chromosome 22q13 and is
composed 23 exons that encode a 1141 amino acid protein.
SREBP-1a regulates all SREBP-responsive genes in both the cholesterol and
fatty acid biosynthetic pathways. SREBP-1c controls the expression of genes
involved in fatty acid synthesis and is involved in the differentiation of adipocytes.
SREBP-1c is also an essential transcription factor downstream of the actions of
insulin at the level of carbohydrate and lipid metabolism. SREBP-2 is the
predominant form of this transcription factor in the liver and it exhibits preference at
controlling the expression of genes involved in cholesterol homeostasis, including all
of the genes encoding the sterol biosynthetic enzymes. In addition SREBP-2
controls expression of the LDL receptor (LDLR) gene.
Regulated expression of the SREBPs is complex in that the effects of sterols
are different on the SREBP-1 gene versus the SREBP-2 gene. High sterols activate
expression of the SREBP-1 gene but do not exert this effect on the SREBP-2 gene.
The sterol-mediated activation of the SREBP-1 gene occurs via the action of the
liver X receptors (LXRs). The LXRs are members of the steroid/thyroid hormone
superfamily of cytosolic ligand binding receptors that migrate to the nucleus upon
ligand binding and regulate gene expression by binding to specific target
sequences. There are two forms of the LXRs: LXRα and LXRβ. The LXRs form
heterodimers with the retinoid X receptors (RXRs) and as such can regulate gene
expression either upon binding oxysterols (e.g. 22R-hydroxycholesterol) or 9-cis-
retinoic acid.
All three SREBPs are proteolytically activated and the proteolysis is controlled
by the level of sterols in the cell. Full-length SREBPs have several domains and are
embedded in the membrane of the endoplasmic reticulum (ER). The N-terminal
domain contains a transcription factor motif of the basic helix-loop-helix (bHLH) type
that is exposed to the cytoplasmic side of the ER. There are two transmembrane
spanning domains followed by a large C-terminal domain also exposed to the
cytosolic side. The C-terminal domain (CTD) interacts with a protein called SREBP
cleavage-activating protein (SCAP). SCAP is a large protein also found in the ER
membrane and contains at least eight transmembrane spans. The C-terminal
portion, which extends into the cytosol, has been shown to interact with the C-
terminal domain of SREBP. This C-terminal region of SCAP contains 4 motifs called
WD40 repeats. The WD40 repeats are required for interaction of SCAP with
SREBP. The regulation of SREBP activity is further controlled within the ER by the
interaction of SCAP with insulin-induced protein-1 and -2 (Insig-1 and Insig-2: see
next paragraph). When cells have sufficient sterol content SREBP and SCAP are
retained in the ER via the SCAP-Insig interaction. The N-terminus of SCAP,
including membrane spans 2–6, resembles HMGR which itself is subject to sterol-
stimulated degradation (see above). This shared motif is called the sterol sensing
domain (SSD) and as a consequence of this domain SCAP functions as the
cholesterol sensor in the protein complex. When cells have sufficient levels of
sterols, SCAP will bind cholesterol which promotes the interaction with Insig and the
entire complex will be maintained in the ER.
 There are two Insig encoding genes identified as INSIG1 and INSIG2. The
INSIG1 gene is located on chromosome 7q36 and is composed of 7 exons that
generate three alternatively spliced mRNAs encoding three isoforms of Insig-1. The
INSIG2 gene is located on chromosome 2q14.2 and is composed of 7 exons that
encode a 225 amino acid protein. The Insig-1 protein was originally isolated in
experiments examining regenerating liver and was subsequently shown to be
dramatically induced in fat tissue in experimental animals at the onset of diet-
induced obesity. INSIG1 gene expression is highest in human liver while INSIG2
gene expression is ubiquitous. The Insig proteins bind to oxysterols which in turn
affects their interactions with SCAP. The major form of human Insig-1 is a 277
amino acids protein and, as indicated, Insig-2 is a 225 amino acid protein. These
two proteins share 59% amino acid identity with the greatest differences being found
in the N- and C-terminal regions. Insig-2 also lacks the 50 amino acids that are
found in the N-terminus of Insig-1. Both Insig proteins can cause ER retention of the
SREBP/SCAP complex. The Insig proteins span the ER membrane six times. It has
been shown that a critical aspartate (D) residue in Insig-1 and Insig-2, found in the
cytosolic loop between membrane spans 4 and 5, is critical for interaction with
SCAP as mutation of this amino acid causes loss of SCAP binding. The third and
fourth transmembrane spans in both Insig proteins are required for interaction with
oxysterols. The Insig-1 gene has been shown to be transcriptionally regulated by
SREBP with the SRE in the Insig-1 gene residing approximately 380bp upstream of
the transcriptional start site. Expression of Insig-1 has also been shown to be
regulated by several members of the nuclear receptor family including PPARδ, PXR
and CAR. The Insig-2 promoter is activated in response to signals downstream of
insulin receptor activation. Nuclear receptors also regulate the expression of the
Insig-2 gene which has been shown to contain two FXR response elements.
In addition to their role in regulating sterol-dependent gene regulation, both Insig
proteins activate sterol-dependent degradation of HMGR. In the presence of the
cholesterol-derived oxysterol, 24,25-dihydrolanosterol, Insig binds to the
transmembrane domain of HMGR. The oxysterol-induced interaction between Insig
and HMGR within the ER membrane allows Insig to recruit the ubiquitin ligase,
gp78, to HMGR resulting in ubiquitination of HMGR and its resultant proteasomal
degradation as described above.
When sterols are scarce, SCAP does not interact with Insig. Under these
conditions the SREBP-SCAP complex migrates to the Golgi where SREBP is
subjected to proteolysis. The cleavage of SREBP is carried out by two distinct
enzymes. The regulated cleavage occurs in the lumenal loop between the 2
transmembrane domains. This cleavage is catalyzed by site-1 protease, S1P (also
known as subtilisin/kexin-isozyme 1, SKI-1). S1P is officially called membrane-
bound transcription factor peptidase, site 1, MBTPS1. The MBTPS1 gene is located
on chromosome 16q24 and is composed of 23 exns that encode a 1052 amino acid
preproprotein. MBTPS1 is a member of the subtilisin-like proprotein convertase 2
family of serine proteases. This family of proteases are responsible for the
processing of proteins that are in the regulated or constitutive branches of the
secretory pathway. The subtilisin-like proprotein convertase 2 family of enzymes are
encoded by nine different genes in humans one of which is the proprotein
convertase subtilisin/kexin type 9 (PCSK9) gene whose encoded enzyme is a recent
target in the treatment of hypercholesteremia (see next section). The function of
SCAP is to positively stimulate S1P-mediated cleavage of SREBP.
The second cleavage, catalyzed by site-2 protease, S2P, occurs in the first
transmembrane span, leading to release of active SREBP. The official name for S2P
is membrane-bound transcription factor peptidase, site 2 (MBTPS2). The MBTPS2
gene is located on the X chromosome (Xp22.12-p22.11) and is composed of 11
exons that encode a 519 amino acid protein. S2P is an intramembrane zinc
metalloprotease. In order for S2P to act on SREBP, site-1 must already have been
cleaved. The result of the S2P cleavage is the release of the N-terminal bHLH motif
into the cytosol. The bHLH domain then migrates to the nucleus where it will
dimerize and form complexes with transcriptional coactivators leading to the
activation of genes containing the SRE motif. To control the level of SREBP-
mediated transcription, the soluble bHLH domain is itself subject to rapid proteolysis.
In addition to the cleavage-activation of SREBP transcriptional activity, S2P is
involved in pathways that regulate cellular responses to endoplasmic reticulum
stress, primarily the unfolded protein response, UPR.

Protease-mediated regulation of SREBP activation. Diagramatic

representation of the interactions between SREBP, SCAP and Insig in the
 membrane of the ER when sterols are high. When sterols are low, SCAP does not
interact with Insig and the SREBP-SCAP complex migrates to the Golgi where the
proteases, S1P and S2P reside. bHLH = basic helix-loop-helix domain. CTD = C-
terminal domain. WD = WD40 domain.

Several proteins whose functions involve sterols also contain the SSD. These
include patched, an important development regulating receptor whose ligand,
hedgehog, is modified by attachment of cholesterol and the Niemann-Pick disease
type C1 (NPC1) protein which is involved in cholesterol transport in the secretory
pathway. NPC1 is one of several genes whose activities, when disrupted, lead to
severe neurological dysfunction.

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Treatment of Hypercholesterolemia
Reductions in circulating cholesterol levels can have profound positive impacts
on cardiovascular disease, particularly on atherosclerosis, as well as other
metabolic disruptions of the vasculature. Control of dietary intake is one of the
easiest and least cost intensive means to achieve reductions in cholesterol. Recent
studies in laboratory rats has demonstrated an additional benefit of reductions in
dietary cholesterol intake. In these animals it was observed that reductions in dietary
cholesterol not only resulted in decreased serum VLDLs and LDLs, and increased
HDLs but DNA synthesis was also shown to be increased in the thymus and spleen.
Upon histological examination of the spleen, thymus and lymph nodes it was found
that there was an increased number of immature cells and enhanced mitotic activity
indicative of enhanced proliferation. These results suggest that a marked reduction
in serum LDLs, induced by reduced cholesterol intake, stimulates enhanced DNA
synthesis and cell proliferation.
Drug treatment to lower plasma lipoproteins and/or cholesterol is primarily
aimed at reducing the risk of atherosclerosis and subsequent coronary artery
disease that exists in patients with elevated circulating lipids. Drug therapy usually is
considered as an option only if non-pharmacologic interventions (altered diet and
exercise) have failed to lower plasma lipids.
Alirocumab (Praluent®), Evolcumab (Repatha®): These drugs are the
newest type of anti-hypercholesterolemia drugs recently approved by the FDA for
use in the US. Both drugs are injectible antibodies that block the function of
proprotein convertase subtilisin/kexin type 9, PCSK9. PCSK9 is serine protease of
the subtilisin-like proprotein convertase 2 family. A major function of PCSK9 is the
endosomal degradation of the LDL receptor (LDLR), thereby reducing the recyling of
the LDLR to the plasma membrane. This effect of PCSK9 leads to a reduced ability
of the liver to remove IDL and LDL from the blood contributing to the potential for
hypercholesterolemia. The potential for the pharmaceutical benefits of the
interference in the activity PCSK9 was recognized by a confluence of several
studies. Patients with a specific form of familial hypercholesterolemia not due to
mutations in the LDLR gene were shown to have severe hypercholesterolemia due
to mutations in the PCSK9 gene resulting in hyperactivity of the enzyme. In addition,
it was found that in certain individuals with low serum LDL levels there was an
association with the inheritance of nonsense mutations in the PCSK9 gene which
result in loss of PCSK9 activity. Hypercholesterolemic patients taking another
cholesterol-lowering drug while simultaneously utilizing either of these new PCSK9
inhibitors saw further reductions in serum LDL levels of betweeen 55% and 77%.
Atorvastatin (Lipitor®), Simvastatin (Zocor®), Lovastatin (Mevacor®):
These drugs are fungal HMG-CoA reductase (HMGR) inhibitors and are members of
the family of drugs referred to as the statins. The net result of treatment is an
increased cellular uptake of LDLs, since the intracellular synthesis of cholesterol is
inhibited and cells are therefore dependent on extracellular sources of cholesterol.
However, since mevalonate (the product of the HMG-CoA reductase reaction) is
required for the synthesis of other important isoprenoid compounds besides
cholesterol, long-term treatments carry some risk of toxicity. A component of the
natural cholesterol lowering supplement, red yeast rice, is in fact a statin-like
compound.
The statins have become recognized as a class of drugs capable of more
pharmacologic benefits than just lowering blood cholesterol levels via their actions
on HMGR. Part of the cardiac benefit of the statins relates to their ability to regulate
the production of S-nitrosylated COX-2. COX-2 is an inducible enzyme involved in
the synthesis of the prostaglandins and thromboxanes as well as the lipoxins and
resolvins. The latter two classes of compounds are anti-inflammatory lipids
discussed in the Lipid-Derived Inflammatory Modulators page. Evidence has shown
that statins activate inducible nitric oxide synthase (iNOS) leading to nitrosylation of
COX-2. The S-nitrosylated COX-2 enzyme produces the lipid compound 15R-
hydroxyeicosatetraenoic acid (15R-HETE) which is then converted via the action of
5-lipoxygenase (5-LOX) to the epimeric lipoxin, 15-epi-LXA4. This latter compound is
the same as the aspirin-triggered lipoxin (ATL) that results from the aspirin-induced
acetylation of COX-2. Therefore, part of the beneficial effects of the statins is
exerted via the actions of the lipoxin family of anti-inflammatory lipids.
Additional anti-inflammatory actions of the statins result from a reduction in the
prenylation of numerous pro-inflammatory modulators. Prenylation refers to the
addition of the 15 carbon farnesyl group or the 20 carbon geranylgeranyl group to
acceptor proteins. The isoprenoid groups are attached to cysteine residues at the
carboxy terminus of proteins in a thioether linkage (C-S-C). A common consensus
sequence at the C-terminus of prenylated proteins has been identified and is
composed of CAAX, where C is cysteine, A is any aliphatic amino acid (except
alanine) and X is the C-terminal amino acid. In addition to numerous prenylated
proteins that contain the CAAX consensus, prenylation is known to occur on
proteins of the RAB family of RAS-related G-proteins. There are at least 60 proteins
in this family that are prenylated at either a CC or CXC element in their C-termini.
The RAB family of proteins are involved in signaling pathways that control
intracellular membrane trafficking. The prenylation of proteins allows them to be
anchored to cell membranes. In addition to cell membrane attachment, prenylation
is known to be important for protein-protein interactions. Thus, inhibition of this post-
translational modification by the statins interferes with the important functions of
many signaling proteins which is manifest by inhibition of inflammatory responses.
Some of the effects on immune function that have been attributed to the statins
are attenuation of autoimmune disease, inhibition of T-cell proliferation, inhibition of
inflammatory co-stimulatory molecule expression, decreases in leukocyte infiltration,
and promotion of a shift in cytokine profiles of helper T-cell types from Th1 to Th2.
Th1 cells are involved in cell-mediated immunity processes, whereas, Th2 cells are
involved in humoral immunity process. The cytokines produced by Th2 cells include
IL-4, IL-5, IL-10 and IL-13 and these trigger B cells to switch to IgE production and
to activate eosinophils.
Nicotinic acid (Niacor® and Niaspan®): Nicotinic acid reduces the plasma
levels of both VLDLs and LDLs by inhibiting hepatic VLDL secretion, as well as
suppressing the flux of FFA release from adipose tissue by inhibiting lipolysis. In
addition, nicotinic administration strongly increases the circulating levels of HDLs.
Patient compliance with nicotinic acid administration is sometimes compromised
because of the unpleasant side-effect of flushing (strong cutaneous vasodilation).
Recent evidence has shown that nicotinic acid binds to and activates the G-protein
coupled receptor identified as GPR109A (also called HM74A or PUMA-G).
GPR109A is a member of the hydroxycarboxylic acid (HCA) receptor family and as
such is now desginated as HCA2 (encoded by the HCAR2 gene). For more detailed
information on the normal biological function of NCA2 (GPR109A) go to the
Bioactive Lipids page. The identity of a receptor to which nicotinic acid binds allows
for the development of new drug therapies that activate the same receptor but that
may lack the negative side-effect of flushing associated with nicotinic acid. Because
of its ability to cause large reductions in circulating levels of cholesterol, nicotinic
acid is used to treat Type II, III, IV and V hyperlipoproteinemias.
 Signaling events initiated in response to β-hydroxybutyrate or nicotinic
acid binding to HCA2 (GPR109A) on adipocytes or macrophages. During
periods of fasting, hepatic ketone synthesis increases and the released β-butyrate
binds to HCA2 on adipocytes triggering activation of the receptor-associated G i-type
G-protein which then inhibits the activity of adenylate cyclase (AC). Inhibition of AC
leads to reduced HSL-mediated release of fatty acids from diacylglycerides.
Nicotinic acid binding to HCA2 on adipocytes also leads to reduced fatty acid
release. The reduced release of adipose tissue fatty acids leads to decreased
synthesis and release of VLDL by the liver. It is this effect of nicotinic acid that
contributes to the antidyslipidemic action of this drug. The HCA2 receptor on
macrophages is also activated by nicotinic acid but this effect contributes to the
undesired side-effets of nicotinic acid therapy. Within macrophages, HCA2
activation results in increased activation of PLA 2 leading to increased arachidonic
acid delivery to COX and increased production of the pro-inflammatory eicosanoids
PGE2 and PGD2. The release of these eicosanoids causes increased cutaneous
vasodilation resulting in the typical flushing and burning pain response to nicotinic
acid therapy.
 Gemfibrozil (Lopid®), Fenofibrate (TriCor®): These compounds (called
fibrates) are derivatives of fibric acid and although used clinically since the 1930's
were only recently discovered to exert some of their lipid-lowering effects via the
activation of peroxisome proliferation. Specifically, the fibrates were found to be
activators of the peroxisome proliferator-activated receptor-α (PPARα) class of
proteins that are classified as nuclear receptor co-activators. The naturally occurring
ligands for PPARα are leukotriene B 4 (LTB4, see the Lipid Synthesis page),
unsaturated fatty acids and oxidized components of VLDLs and LDLs. The PPARs
interact with another receptor family called the retinoid X receptors (RXRs) that bind
9-cis-retinoic acid. Activation of PPARs results in modulation of the expression of
genes involved in lipid metabolism. In addition the PPARs modulate carbohydrate
metabolism and adipose tissue differentiation. Fibrates result in the activation of
PPARα in liver and muscle. In the liver this leads to increased peroxisomal β-
oxidation of fatty acids, thereby decreasing the liver's secretion of triacylglycerol-
and cholesterol-rich VLDLs, as well as increased clearance of chylomicron
remnants, increased levels of HDLs and increased lipoprotein lipase activity which in
turn promotes rapid VLDL turnover.
Cholestyramine or colestipol (resins): These compounds are nonabsorbable
resins that bind bile acids which are then not reabsorbed by the liver but excreted.
The drop in hepatic reabsorption of bile acids releases a feedback inhibitory
mechanism that had been inhibiting bile acid synthesis. As a result, a greater
amount of cholesterol is converted to bile acids to maintain a steady level in
circulation. Additionally, the synthesis of LDL receptors increases to allow increased
cholesterol uptake for bile acid synthesis, and the overall effect is a reduction in
plasma cholesterol. This treatment is ineffective in homozygous FH patients, since
they are completely deficient in LDL receptors.
Ezetimibe: This drug is sold under the trade names Zetia® or Ezetrol® and is
also combined with the statin drug simvastatin and sold as Vytorin® or Inegy®.
Ezetimibe functions to reduce intestinal absorption of cholesterol, thus effecting a
reduction in circulating cholesterol. The drug functions by inhibiting the intestinal
brush border transporter involved in absorption of cholesterol. This transporter is
known as Niemann-Pick type C1-like 1 (NPC1L1). NPC1L1 is also highly expressed
in human liver. The hepatic function of NPC1L1 is presumed to limit excessive
biliary cholesterol loss. NPC1L1-dependent sterol uptake is regulated by cellular
cholesterol content. In addition to the cholesterol lowering effects that result from
inhibition of NPC1L1, its inhibition has been shown to have beneficial effects on
components of the metabolic syndrome, such as obesity, insulin resistance, and
fatty liver, in addition to atherosclerosis. Ezetimibe is usually prescribed for patients
who cannot tolerate a statin drug or a high dose statin regimen. There is some
controversy as to the efficacy of ezetimibe at lowering serum cholesterol and
reducing the production of fatty plaques on arterial walls. The combination drug of
ezetimibe and simvastatin has shown efficacy equal to or slightly greater than
atorvastatin (Lipitor®) alone at reducing circulating cholesterol levels.
New Approaches: Numerous epidemiological and clinical studies over the past
10 years have demonstrated a direct correlation between the circulating levels of
 HDL cholesterol (most often abbreviated HDL-c) and a reduction in the potential for
atherosclerosis and coronary heart disease (CHD). Individuals with levels of HDL
above 50mg/dL are several time less likely to experience CHD than individuals with
levels below 40mg/dL. In addition, clinical studies in which apolipoprotein A-I (apoA-
I), the predominant protein component of HDL-c) or reconstituted HDLs are infused
into patients raises circulating HDL levels and reduces the incidence of CHD. Thus,
there is precedence for therapies aimed at raising HDL levels in the treatment and
prevention of atherosclerosis and CHD. Unfortunately current therapies only
modestly elevate HDL levels. Both the statins and the fibrates have only been
shown to increase HDL levels between 5–20% and niacin is poorly tolerated in
many patients. Therefore, alternative strategies aimed at increasing HDL levels are
being tested. Cholesterol ester transfer protein (CETP) is secreted primarily from the
liver and plays a critical role in HDL metabolism by facilitating the exchange of
cholesteryl esters (CE) from HDL for triglycerides (TG) in apoB containing
lipoproteins, such as LDL and VLDL. The activity of CETP directly lowers the
cholesterol levels of HDLs and enhances HDL catabolism by providing HDLs with
the TG substrate of hepatic lipase. Thus, CETP plays a critical role in the regulation
of circulating levels of HDL, LDL, and apoA-I. It has also been shown that in mice
naturally lacking CETP most of their cholesterol is found in HDL and these mice are
relatively resistant to atherosclerosis. The potential for the therapeutic use of CETP
inhibitors in humans was first suggested when it was discovered in 1985 that a small
population of Japanese had an inborn error in the CETP gene leading to
hyperalphalipoproteinemia and very high HDL levels. To date three CETP inhibitors
have been used in clinical trials. These compounds are anacetrapib, torcetrapib, and
dalcetrapib. Although torcetrapib is a potent inhibitor of CETP, its use has been
discontinued due to increased negative cardiovascular events and death rates in
test subjects. Treatment with dalcetrapib results in increases in HDL (19–37%) and
a modest decrease (≈6%) in LDL levels. Treatment with anacetrapib results in a
significant increase in both HDL (≈130%) and LDL (≈40%). Anacetrapib is currently
in phase III clinical studies.
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Last modified: May 26, 2017