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Cholesterol: Synthesis, Metabolism,

and Regulation
! Biochemistry Topics , ! Lipid Metabolism

Table of Contents 
 1. Introduction to Cholesterol Metabolism
2. Biosynthesis of Cholesterol
2.0.1. HMG-CoA Synthesis
2.0.2. Mevalonate Synthesis
2.0.3. Isopentenylpyrophosphate (IPP) Synthesis
2.0.4. Squalene Synthesis
2.0.5. Squalene to Cholesterol
3. Cytochrome P450 Enzymes in Cholesterol Metabolism
4. Important Isoprenoids from Intermediates of Cholesterol Synthesis
4.1. Dolichol Phosphate Synthesis
4.2. Coenzyme Q (Ubiquinone) Synthesis
4.3. Heme a (heme A) Synthesis
5. Regulating Cholesterol Synthesis
6. Proteolytic Regulation of HMG-CoA Reductase
7. The Utilization of Cholesterol
8. Regulation of Cellular Sterol Content
9. Treatment of Hypercholesterolemia
9.1. Potential Future Therapies for Hyperlipidemia

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.
 Structure of cholesterol

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 initial steps in the pathway of
cholesterol biosynthesis are collectively called the mevalonate pathway which itself
culminates with the synthesis of the isoprenoid molecule, isopentenyl
pyrophosphate (IPP).

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 modi!cation of some proteins.

Pathway for the movement of acetyl-CoA units from within

the mitochondrion to the cytoplasm. Under high energy
charge mitochondrial acetyl-CoA and citrate accumulate due to
allosteric inhibition of the TCA cycle. Due to accumulating acetyl-
CoA, pyruvate carboxylase is highly activated allowing for
continued synthesis of oxaloacetate ensuring the eventual
synthesis of citrate. 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. Transport across the inner
mitochondrial membrane requires a heterotetrameric transport
complex (mitochondrial pyruvate carrier) consisting of the MPC1
gene and MPC2 gene encoded proteins. Note that the
cytoplasmic malic enzyme (encoded by the ME1 gene) catalyzed
reaction generates NADPH which can be used for reductive
biosynthetic reactions such as those of fatty acid, cholesterol
synthesis, and phospholipid biosythesis.
The process of cholesterol synthesis can be considered to be composed of !ve
major steps where the reactions that culminate in the synthesis of isopentenyl
pyrophosphate, and its isomeric form dimethylallyl pyrophosphate, are commonly
referred to as the mevlonate pathway:

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)
4. IPP molecules are 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 mevalonate 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 di"use 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 di"erent 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 isopentenyl pyrophosphate (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
(DMAPP) 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 DMAPP 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 !ve alternatively spliced mRNAs that, together, encode three
di"erent isoforms of the enzyme.

The synthesis of squalene, from FPP, represents the !rst cholesterol-speci!c 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 di"erent FDFT1-encoded mRNAs collectively
synthesize !ve di"erent isoforms of farnesyl-diphosphate farnesyltransferase 1.

Squalene to Cholesterol
Squalene then undergoes a two step cyclization to yield lanosterol. The !rst 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 di"erent 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. De!ciency 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.

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 below).

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. De!ciency 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 su"ering 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 12α-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 ferrodoxin 1
(also called adrenodoxin) and ferrodoxin reductase (also called 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. De!ciencies 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 identi!ed 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 e"ect 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
e#ciently 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 !broblast growth factor receptor 2 (FGFR2) gene.

Important Isoprenoids from Intermediates of

Cholesterol Synthesis
Dolichol Phosphate Synthesis
Dolichol phosphate is a polyisoprenoid compound synthesized from the isoprenoid
intermediates of the de novo cholesterol biosynthesis pathway. The function of
dolichol phosphate is to serve as the foundation for the synthesis of the precursor
carbohydrate structure, termed the lipid-linked oligosaccharide, LLO ( also referred
to as the en bloc oligosacchariode), required for the attachment of carbohydrate to
asparagine residues in N-linked glycoproteins.

As indicated in the Figure above showing the pathway of cholesterol biosynthesis a

molecule of geranylpyrophosphate (GPP) and a molecule of
isopentenylpyrophosphate (IPP) are condensed into farnesylpyrophosphate (FPP)
through the action of the farnesyl diphosphate synthase enzyme which is encoded
by the FDPS gene. Through the action of the ER-localized enzyme, dehydrodolichyl
diphosphate synthase (encoded by the DHDDS gene), farnesylpyrophosphate is
elongated via the sequential head-to-tail addition of multiple
isopentenylpyrophosphate groups in a reaction referred to as cis-prenylation. The
number of IPP substrates added ultimately determines the overall number of
isoprene units in dolichol which in humans ranges from 17 to 21. The DHDDS gene
is located on chromosome 1p36.11 and is composed of 10 exons that generate !ve
alternatively spliced mRNAs each of which encode a distince protein isoform. The
product(s) of the DHDDS reaction is referred to as a polyprenolpyrophosphate. The
pyrophosphate is removed by an as yet uncharacterized enzyme activity that may
be either a polyprenol pyrophosphate phosphatase or a polyprenol phosphatase
resulting in the formation of a polyprenol.

The resultant polyprenol(s) is a substrate for steroid 5-α reductase 3 (also called
polyprenol reductase) which is encoded by the SRD5A3 gene. Steroid 5-α reductase
3 belongs to the polyprenol reductase sufamily of the steroid 5-α reductase family.
The SRD5A3 encoded enzyme reduces the carbon-carbon double bond closest to
the hydroxyl end of the polyprenol generating dolichol. In addition to participating
in the synthesis of dolichol the SRD5A3 encoded enzyme synthesizes 5-α-
dihydrotestosterone from testosterone. The SRD5A3 gene is located on
chromosome 4q12 and is composed of 6 exons that encode a 318 amino acid
protein. Mutations in the SRD5A3 gene are associated with the congenital disorder
of glycosylation (CDG) identi!ed as CDG-1q (SRD5A3-CDG).

Dolichol phosphate is then synthesized from dolichol through the action of the ER-
localized enzyme dolichol kinase. The phosphate donor for dolichol kinase is CTP
and not ATP as is the case for most kinases. Dolichol kinase is encoded by the DOLK
gene which is located on chromosome 9q34.11 which is an intronless gene that
encodes a 538 amino acid protein. Mutations of the DOLK gene are assoicated with
the CDG identi!ed as CDG-1m (DOLK-CDG).
Pathway of dolichol phosphate biosynthesis. Synthesis of
dolichol phosphate begins with the farnesylpyrophosphate
synthesized in the !rst part of the cholesterol biosynthesis
pathway. Farnesylpyrophosphate is elongated through
sequential head-to-tail condensation reactions with
 isopentenylpyrophosphate catalyzed by dehydrodolichyl
diphosphate synthase (DHDDS). This initial process generates
polyisoprenoidpyrophosphate compounds that have varying
numbers of isoprene units ranging from 17–21 in humans. The
pyrophosphate is removed, by incompletely characterized
enzymatic activities, forming polyprenol compounds. Polyprenols
are then reduced to dolichols through the action of steroid 5α-
reductase 3 (SRD5A3). The resultant dolichol is then
phosphorylated on the alcohol forming dolichol phosphate
through the action of CTP-dependent dolichol kinase (DOLK).

Coenzyme Q (Ubiquinone) Synthesis

Coenzyme Q (ubiquinone) is a red-ox active molecule that is composed of a
benzoquinone ring conjugated to a polyisoprenoid tail that is of variable length in
di"erent species and organisms. In humans the polyisoprenoid tail consists of 10
isoprenoid units which impart the common name for the molecule as CoQ10. A
minor amount of ubiquinone in humans contains 9 isoprenoid units. In undergoing
reduction and oxidation reaction the electrons are accepted and donated from
benzoquinone ring. The polyisoprenoid tail of ubiquinone serves to anchor the
molcule in the membrane.

Structure of human coenzyme Q10 (ubiquinone)

The complete pathway for the synthesis of ubiquinone in eukaryotes has been
worked out in yeasts and the round worm, Caenorhabditis elegans. In humans,
homologues of all of the yeast genes have been found. The initial steps in the
synthesis of ubiquinone involve the formation of the polyisoprenoid tail. In human
tissues a molecule of farnesy pyrophosphate and a molecule of isopentenyl
pyrophosphate are condensed to form all trans-decaprenyl diphosphate. This
reaction is catalyzed by the heterotetrameric enzyme identi!ed as decaprenyl
diphosphate synthase. The two di"erent subunits of the enzyme are encoded by
the PDSS1 and PDSS2 genes. The PDSS1 gene is located on chromosome 10p12.1
and is composed of 14 exons that generate three alternatively spliced mRNAs each
of which encode distinct protein isoforms. The PDSS2 gene is located on
chromosome 6q21 and is composed of 11 exons that encode a protein of 399
amino acids. The remainder of the genes involved in human ubiquinone synthesis
all have the designation COQ.

Following synthesis of the decaprenyl molecule, the enzyme, 4-hydroxybenzoate

polyprenyltransferase (encoded by the COQ2 gene), catalyzes covalent attachment
of the decaprenyl diphosphate to the aromatic ring of 4-hydroxybenzoate (para-
hydroxybenzoate) forming 3-decaprenyl-4-hydroxybenzoic acid. The COQ2 encoded
protein is localized to the mitochondria. The COQ2 gene is located on chromosome
4q22.22–q21.23 and is composed of 17 exons that encode a protein of 421 amino
acids. Mutations in the COQ2 gene are associated with a form of mitochondrial
encephalomyopathy as well as a COQ2 nephropathy.

After the attachment of the decaprenyl group the aromatic ring undergoes a series
of modi!cations. The !rst modi!cation is a hydroxylation reaction at carbon 5 of the
benzene ring. This hydroxylation is catalyzed by the FAD-dependent
monooxygenase encoded by the COQ6 gene. The COQ6 gene is located on
chromosome 14q24.3 and is composed of 15 exons that generate two alternatively
spliced mRNAs each encoding a distinct protein isoform. In the next reaction the
newly attached hydroxyl group undergoes an O-methylation reaction catalyzed by
the mitochondrial SAM-dependent O-methyltransferase encoded by the COQ3 gene.
The COQ3 gene is located on chromosome 6q16.2 and is composed of 9 exons that
encode a 369 amino acid protein.

The next reaction involves decarboxylation of the carboxylic acid group attached to
carbon 1 of the benzene ring leaving a hydroxyl group. The decarboxylation reaction
is catalyzed by an as yet uncharacterized enzyme. These three reactions result in
the formation of 2-methoxy-6-decaprenylphenol.

In the next reaction, carbon 2 of the benzene ring is methylated. The C-methylation
reaction is catalyzed by the mitochondrial SAM-dependent enzyme identi!ed as 2-
methoxy-6-polyprenyl-1,4-benzoquinol methylase. This methylase is encoded by the
COQ5 gene which is located on chromosome 12q24.31 and is composed of 8 exons
that encode a 327 amino acid protein.

The next reaction involves the hydroxylation of carbon 6 of the benzene ring. This
hydroxylation is catalyzed by 5-demethoxyubiquinone hydroxylase which is
encoded by the COQ7 gene. The COQ7 gene is located on chromosome 16p12.3 and
is composed of 8 exons that generate two alternatively spliced mRNAs both of
which encode distinct protein isoforms.

The !nal reaction in ubiquinone synthesis is a SAM-dependent methylation of the

newly added hydroxyl group. This last reaction is catalyzed the COQ3 encoded O-
methyltransferase.

Heme a (heme A) Synthesis

Heme a (heme A) is an essential component of the oxidative
phosphorylation pathway by serving as the prosthetic group for
cytochrome aa3 (also called cytochrome c oxidase) of complex IV. Cytochrome aa3 is
so-called due to the presence of two distinct heme a prosthetic groups with
heme a being the direct electron donor in the complex IV catalyzed reduction of
O2 to H2O. The heme a3 prosthetic group constitutes part of the copper-dependent
active site of complex IV.

Heme a is synthesized from heme b (iron protoporphryin IX) through a series of

reactions that convert the methyl side group on carbon 8 (C8) of the porphyrin
molecule into a formyl group along with conversion of the vinyl group at position C2
to hydroxyethylfarnesyl with the isoprenoid farnesyl pyrophosphate as the
substrate. The transfer of the farnesyl group to the C2 vinyl group is catalyzed by
the enzyme identi!ed as heme A:farnesyltransferase cytochrome c oxidase
assembly factor (also called protoheme IX farnesyltransferase). This enzyme, which
is localized to the inner mitochondrial membrane, is encoded by the COX10 gene.
The COX10 gene is located on chromosome 17p12 and is composed of 7 exons that
encode a 443 amino acid protein. The addition of the farnesyl group to
heme a generates the heme identi!ed as heme o (heme O). Heme o is then
converted to heme a through a series of reactions the converts the C8 methyl group
into a formyl group. The conversion of heme o to heme a is catalyzed by the enzyme
identi!ed as cytochrome c oxidase assembly protein COX15 homolog which is
encoded by the COX15 gene. Like the COX10 encoded protein, the COX15 encoded
protein is localized to the inner mitochondrial membrane. The COX15 gene is
located on chromosome 10q24.2 and is composed of 10 exons that generate !ve
alternatively spliced mRNAs that collectively encode four distinct protein isoforms.

Structure of heme A

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 con$icts with the o#cial 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 !rst 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 polyubiquitylation of
HMGR and its degradation in the proteasome (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) a"ect the transcription of the HMGR gene is
described below under regulation of sterol content.

Regulation of HMGR through covalent modi!cation occurs as a result of

phosphorylation and dephosphorylation. The enzyme is most active in its
unmodi!ed form. Phosphorylation of the enzyme decreases its activity. HMGR is
phosphorylated by AMP-activated protein kinase, AMPK. 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 !rst identi!ed 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
2+
of AMPK in response to increases in intracellular Ca 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 modi!cation. 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. Dephosphorylation of HMGR, returning it to
the more active state, is e"ected via the activity of protein
phosphatases of the 2A family (PP2A). Functional PP2A exists in
two distinct catalytic isoforms encoded by two genes identi!ed as
PPP2CA and PPP2CB. The two basic isoforms of PP2A are a
heterodimeric core enzyme and a heterotrimeric holoenzyme.
The PP2A core enzyme consists of a sca"old subunit (originally
termed the A subunit) and a catalytic subunit (the C subunits).
The catalytic α subunit is encoded by the PPP2CA gene and the
catalytic β subunit is encoded by the PPP2CB gene. The sca"old α
subunit is encoded by the PPP2R1A gene and the β subunit by
the PPP2R1B gene. The PP2A core enzyme interacts with a
 variable regulatory subunit to assemble into a holoenzyme. The
PP2A regulatory subunits comprise four families (originally
identi!ed as the B subunits) each of which consists of multiple
isoforms that are encoded by di"erent genes. There are currently
15 di"erent PP2A B regulatory subunit genes expressed in
humans. The major function of the PP2A regulatory subunits is to
target phosphorylated substrate proteins to the phosphatase
activity of the PP2A catalytic subunits. PPP2R represents one of
the 15 di"erent PP2A regulatory subunits. Hormones such as
glucagon and epinephrine negatively a"ect cholesterol
biosynthesis by increasing the activity of the speci!c regulatory
subunits of the PP2A family enzymes. PKA-mediated
phosphorylation of a PP2A regulatory subunit (PPP2R) results in
release of PP2A from HMGR preventing it from being
dephosphorylated. Opposing the e"ects 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 PP2A
(PPP2R in Figure) leading to an increase in release of PP2A from HMGR. This
prevents PP2A from removing phosphates from HMGR preventing its reactivation.
The large family of protein phosphatase regulatory subunits regulate and/or inhibit
the activity of numerous phosphatases including members of the PP1, PP2A, and
PP2C families. In addition to PP2A phosphatases that remove phosphates from
AMPK and HMGR, phosphatases of the protein phosphatase 2C (PP2C) family also
remove phosphates from AMPK. When these regulatory subunits are
phosphorylated by PKA the activity of the associated phosphatases is reduced which
results in AMPK remaining 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. On the other hand, insulin leads to a decrease in cAMP, which in turn
activates cholesterol synthesis.

The ability of insulin to stimulate, and glucagon to inhibit, HMGR activity is

consistent with the e"ects 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.

Proteolytic Regulation of HMG-CoA Reductase

The stability of HMGR is regulated as the rate of $ux through the mevalonate
synthesis pathway changes. When the $ux is high the rate of HMGR degradation is
also high. When the $ux 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 proteasome, a multiprotein complex dedicated to protein degradation. The
primary signal directing proteins to the proteasome 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 proteasome. 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.

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 LDL. The liver synthesizes VLDL and these are converted to LDL
through the action of endothelial cell-associated lipoprotein lipase. Cholesterol
found in plasma membranes can be extracted by HDL and esteri!ed by the HDL-
associated enzyme lecithin-cholesterol acyltransferase, LCAT. The cholesterol
acquired from peripheral tissues by HDL can then be transferred to VLDL and LDL
via the action of cholesteryl ester transfer protein (CETP) which is associated with
HDL. Reverse cholesterol transport allows peripheral cholesterol to be returned to
the liver in LDL. Ultimately, cholesterol is excreted in the bile as free cholesterol or
as bile salts following conversion to bile acids in the liver.

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 a"ects 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 identi!ed 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
identi!ed as SREBP-1a and SREBP-1c/ADD1 (ADD1 is adipocyte di"erentiation-1) as
a consequence of alternative transcriptional start sites resulting in the utilization of
di"erent !rst 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 di"erentiation 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 e"ects of sterols are
di"erent on the SREBP-1 gene versus the SREBP-2 gene. High sterols activate
expression of the SREBP-1 gene but do not exert this e"ect 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 speci!c 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 su#cient 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 su#cient 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 identi!ed 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 a"ects 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 di"erences 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 two transmembrane
domains. This cleavage is catalyzed by site-1 protease, S1P [also known as
subtilisin/kexin-isozyme 1 (SKI-1) and as proprotein convertase subtilisin/kexin type
8 (PCSK8)]. S1P is o#cially called membrane-bound transcription factor peptidase,
site 1, MBTPS1. The MBTPS1 gene is located on chromosome 16q23.3–q24.1 and is
composed of 24 exons 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 di"erent 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 !rst
transmembrane span, leading to release of active SREBP. The o#cial name for S2P
is membrane-bound transcription factor peptidase, site 2 (MBTPS2). The MBTPS2
gene is located on the X chromosome (Xp22.12) 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 modi!ed 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.

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 bene!t of reductions in dietary
cholesterol intake. In these animals it was observed that reductions in dietary
cholesterol not only resulted in decreased serum VLDL and LDL, and increased HDL
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 LDL, 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 e"ect 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 bene!ts of the
interference in the activity PCSK9 was recognized by a con$uence of several studies.
Patients with a speci!c 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 LDL, 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 bene!ts than just lowering blood cholesterol levels via their actions
on HMGR. Part of the cardiac bene!t 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-in$ammatory lipids
discussed in the Lipid-Derived In$ammatory 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 bene!cial e"ects of the statins is
exerted via the actions of the lipoxin family of anti-in$ammatory lipids.

Additional anti-in$ammatory actions of the statins result from a reduction in

the prenylation of numerous pro-in$ammatory modulators. Prenylation refers to
the addition of the 15 carbon farnesyl group or the 20 carbon geranylgeranyl group
to acceptor proteins. The isoprenoid groups are attached to cysteine residues at the
carboxy terminus of proteins in a thioether linkage (C-S-C). A common consensus
sequence at the C-terminus of prenylated proteins has been identi!ed and is
composed of CAAX, where C is cysteine, A is any aliphatic amino acid (except
alanine) and X is the C-terminal amino acid. In addition to numerous prenylated
proteins that contain the CAAX consensus, prenylation is known to occur on
proteins of the RAB family of RAS-related G-proteins. There are at least 60 proteins
in this family that are prenylated at either a CC or CXC element in their C-termini.
The RAB family of proteins are involved in signaling pathways that control
intracellular membrane tra#cking. The prenylation of proteins allows them to be
anchored to cell membranes. In addition to cell membrane attachment, prenylation
is known to be important for protein-protein interactions. Thus, inhibition of this
post-translational modi!cation by the statins interferes with the important functions
of many signaling proteins which is manifest by inhibition of in$ammatory
responses.

Some of the e"ects on immune function that have been attributed to the statins are
attenuation of autoimmune disease, inhibition of T-cell proliferation, inhibition of
in$ammatory co-stimulatory molecule expression, decreases in leukocyte
in!ltration, and promotion of a shift in cytokine pro!les 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 VLDL and LDL by inhibiting hepatic VLDL secretion, as well as suppressing
the $ux of FFA release from adipose tissue by inhibiting lipolysis. In addition,
nicotinic administration strongly increases the circulating levels of HDL. Patient
compliance with nicotinic acid administration is sometimes compromised because
of the unpleasant side-e"ect of $ushing (strong cutaneous vasodilation). Recent
evidence has shown that nicotinic acid binds to and activates the G-protein coupled
receptor identi!ed 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-e"ect of $ushing 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 Gi-
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
e"ect 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 e"ect contributes to the
undesired side-e"ects of nicotinic acid therapy. Within
macrophages, HCA2 activation results in increased activation of
PLA2 leading to increased arachidonic acid delivery to COX and
increased production of the pro-in$ammatory eicosanoids PGE2
and PGD2. The release of these eicosanoids causes increased
 cutaneous vasodilation resulting in the typical $ushing and
burning pain response to nicotinic acid therapy.

Gem!brozil (Lopid®), Feno!brate (TriCor®): These compounds (called !brates)

are derivatives of !bric acid and although used clinically since the 1930’s were only
recently discovered to exert some of their lipid-lowering e"ects via the activation of
peroxisome proliferation. Speci!cally, the !brates were found to be activators of
the peroxisome proliferator-activated receptor-α (PPARα) class of proteins that are
classi!ed as nuclear receptor co-activators. The naturally occurring ligands for
PPARα are leukotriene B4 (LTB4, see the Eicosanoid Metabolism page), unsaturated
fatty acids and oxidized components of VLDL and LDL. 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 di"erentiation. 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 VLDL,
as well as increased clearance of chylomicron remnants, increased levels of HDL
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 e"ect is a reduction in
plasma cholesterol. This treatment is ine"ective in homozygous FH patients, since
they are completely de!cient 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 e"ecting 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 e"ects that result from
inhibition of NPC1L1, its inhibition has been shown to have bene!cial e"ects 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 e#cacy 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 e#cacy equal to or slightly greater than
atorvastatin (Lipitor®) alone at reducing circulating cholesterol levels.

Lomitapide: Certain patient populations, especially individuals that are

homozygous for mutations in the LDL receptor are not e"ectively treated with drugs
such as alirocumab and statins. Two recent drugs, that were designed to target liver
lipoprotein homeostasis, have been approved for use in humans. One drug,
lomitapide (Juxtapid®), is a small molecule inhibitor of the microsomal triglyceride
transfer protein (MTTP; also referred to as MTP). MTTP is a heterodimeric complex
composed of a large subunit (encoded by the MTTP gene) and a small subunit which
is a member of the protein disul!de isomerase (PDI) family of enzymes that are
involved in protein folding. The MTTP complex is required for the incorporation of
apoB-48 into chylomicrons in the intestines and apoB-100 into VLDL by the liver.
Lomitapide has been shown to reduce circulating LDL in homozygous FH patients by
up to 50%.

Mipomersen: The drug mipomersen (Kynamro®) is an anti-sense oligonucleotide

(ASO) that targets the apoB mRNA in the liver, thereby resulting in reduced
synthesis of the apoB-100 protein. Since apoB-100 is required for VLDL assembly in
the liver there is reduced VLDL secretion by the liver. In homozygous LDL receptor
gene mutation patients with familial hypercholesterolemia (FH) who take
mipomersen there was an observed reduction of circulating LDL by approximately
25%.

Bempedoic Acid: Bempedoic acid is a dicarboxylic acid that was demonstrated to

inhibit fatty acid and cholesterol synthesis in experimental animals and these e"ects
were correlated to reductions in plasma triglyceride and lipoprotein levels.
Bempedoic acid is a pro-drug that is converted exclusively in the liver to its active
CoA-derivative, bempedoyl-CoA. The CoA addition to bempedoic acid is catalyzed by
very long-chain acyl-CoA synthetase-1 (ACSVL1) which is encoded by
the SLC27A2 gene (see the Fatty Acid Oxidation page). The SLC27A2 gene id highly
expressed in the liver but is not expressed in adipose tissue, the intestines nor in
skeletal muscle. The conversion of bempedoic acid to its CoA derivative is required
for its ability to suppress lipid synthesis and to also stimulate of mitochondrial fatty
acid β-oxidation. One advantage of bempedoic acid over statins in the treatment of
hypercholesterolemia is that the lack of SLC27A2 expression in skeletal muscle
would prevent any adverse side e"ects in that tissue. The inhibition of muscle
cholesterol systhesis by statins is a cause of the associated myotoxicity of that class
of drug. Indeed, during clinical trials of bempedoic acid there was an absence of any
muscle related symptoms. The US FDA approved the use of orally administered
bempedoic acid alone (Nexletol™) or in combination with ezetimibe (Nexlizet™) in
February of 2020.

Potential Future Therapies for Hyperlipidemia

Several other potential targets have been identi!ed that may prove useful for
pharmacological intervention of hyperlipidemias and hypercholesterolemias. The
apolipoprotein apoC-III (encoded by the APOC3 gene) inhibits the activity of
endothelial lipoprotein lipase (LPL) as well as hepatic lipase. Individuals harboring
loss-of-function mutations in the APOC3 gene have signi!cantly reduced levels of
circulating triglycerides suggeting that targeting this protein may be an e"ective
treatment of hypertriglyceridemias. An ASO approach to knocking down the
circulating level of apoC-III is currently in phase 2 clinical trials.

Humans express two additional LPL inhibitors encoded by the ANGPTL3 and
ANGPTL4 genes. These genes encode proteins of the angiopoietin-like family that
not only inhibit LPL but also inhibit endothelial lipase (encoded by the LIPG gene).
Mutations in the ANGPTL3 gene are associated with reduced levels of circulating
triglycerides in a disorder called familial combined hypolipidemia. These genetic
observations suggest that blocking the function of ANGPTL3 may be useful in
treating hypertriglyceridemias.However, given that ANGPTL3 activity is positively
correlated to increased levels of phospholipid-rich HDL, which is highly active at
cholesterol transport, there may be risks to blocking ANGPTL3 activity entirely.
Indeed, humans with homozygous loss-of-function mutations in the ANGPTL3 gene
have low serum levels of not only triglycerides and LDL but also low levels of HDL.
Phase 3 clinical trials of a monoclonal antibody targeting ANGPTL3 (evinacumab)
have shown promise at lowering circulating levels of LDL in homozygous familial
hypercholesterolemia (designated (HoFH) patients. Patients were administered the
monoclonal antibody over a period of 24 weeks and showed an average reduction
in LDL cholesterol (LDL-c) of 49%.

Numerous epidemiological and clinical studies over the past 15 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 demonstrated that when apolipoprotein A-I
(apoA-I; the predominant protein component of HDL-c), or reconstituted HDL are
infused into patients there was an increase in the level of circulating HDL and
reduced 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 !brates 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. IDL, and VLDL. The activity of CETP
directly lowers the cholesterol levels of HDL and enhances HDL catabolism by
providing HDL 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 !rst 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
four CETP inhibitors have been used in clinical trials. These compounds are
anacetrapib, torcetrapib, dalcetrapib, and evocetrapib. 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. Studies with dalcetrapib and
evocetrapib showed no real bene!ts and the trials were terminated. Treatment with
anacetrapib results in a signi!cant increase in both HDL (104%) and LDL (17%).
Anacetrapib trials went to completion and it is also being tested in conjunction with
statin drugs.

Biochemistry Topics

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