Introduction to Cholesterol MetabolismBiosynthesis of
CholesterolRegulation of Cholesterol SynthesisProteolytic
Regulation of HMG-CoA ReductaseUtilization of CholesterolCytochrome
P450 Enzymes in Cholesterol MetabolismRegulation of Cellular Sterol
ContentSerum Cholesterol ValuesTreatment of
Hypercholesterolemia
Bile Acid Synthesis and Metabolism
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Introduction to Cholesterol MetabolismCholesterol 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 and bile acids. 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.The synthesis and
utilization of cholesterol must be tightly regulated in order to
prevent over-accumulation and abnormal deposition within the body.
Of particular importance clinically 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
back to the top
Biosynthesis of CholesterolSlightly 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.
Cholesterol synthesis occurs in the cytoplasm and microsomes (ER)
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 (ADH3). 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 for use in lipid and cholesterol
biosynthesis. 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). SLC16A1 is the pyruvate transporter
(also called the monocarboxylic acid transporter).The process of
cholesterol synthesis has five major steps:1. Acetyl-CoAs are
converted to 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) 2. HMG-CoA is
converted to mevalonate3. Mevalonate is converted to the isoprene
based molecule, isopentenyl pyrophosphate (IPP), with the
concomitant loss of CO24. IPP is converted to squalene5. Squalene
is converted to cholesterol.
Pathway of cholesterol biosynthesis. Synthesis begins with the
transport of acetyl-CoA from the mitochondrion to the cytosol. The
rate limiting step 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. Place mouse over intermediate names to see
structure.
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
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
HMG-CoA synthase.HMG-CoA is 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. Mevalonate is then
activated by two successive phosphorylations (catalyzed by
mevalonate kinase, and phosphomevalonate kinase), yielding
5-pyrophosphomevalonate. In humans, mevalonate kinase resides in
the cytosol indicating that not all the reactions of cholesterol
synthesis are catalyzed by membrane-associated enzymes as
originally described. After phosphorylation, an ATP-dependent
decarboxylation yields isopentenyl pyrophosphate, IPP, an activated
isoprenoid molecule. Isopentenyl pyrophosphate is in equilibrium
with its isomer, dimethylallyl pyrophosphate, DMPP. 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. Finally, the NADPH-requiring
enzyme, squalene synthase catalyzes the head-to-tail condensation
of two molecules of FPP, yielding squalene. Like HMGR, squalene
synthase is tightly associated with the ER. Squalene undergoes a
two step cyclization to yield lanosterol. The first reaction is
catalyzed by squalene monooxygenase. This enzyme uses NADPH as a
cofactor to introduce molecular oxygen as an epoxide at the 2,3
position of squalene. Through a series of 19 additional reactions,
lanosterol is converted to cholesterol.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 membrane. 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.back to the top
Regulating Cholesterol SynthesisNormal 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 (150200 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
acyl-CoA:cholesterol acyltransferase, ACAT3. 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 2 enzymes. The primary kinase
sensitive to rising AMP levels is LKB1. 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 (CaMKK). CaMKK
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 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 2 enzymes: LKB1 and CaMKK. Hormones such as
glucagon and epinephrine negatively affect cholesterol biosynthesis
by increasing the activity of the inhibitor of phosphoprotein
phosphatase inhibitor-1, PPI-1. Conversely, insulin stimulates the
removal of phosphates and, thereby, activates HMGR activity.
Additional regulation of HMGR occurs through an inhibition of its'
activity as well as of its' synthesis by elevation in intracellular
cholesterol 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 phosphoprotein
phosphatase inhibitor-1 (PPI-1) leading to an increase in its'
activity. PPI-1 can inhibit the activity of numerous phosphatases
including protein phosphatase 2C (PP2C) and PP2A (also called HMGR
phosphatase) which remove phosphates from AMPK and HMGR,
respectively. 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. back to the
top
Proteolytic Regulation of HMG-CoA ReductaseThe 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.back to the top
The Utilization of CholesterolCholesterol 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 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
(apo-D) 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.back to the top
Cytochrome P450 Enzymes in Cholesterol MetabolismCytochrome 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
4-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 7-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 7-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 7-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-14-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 14-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.back to the top
Regulation of Cellular Sterol ContentThe 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. It
turns out that there are 2 distinct SREBP genes, SREBP-1 and
SREBP-2. In addition, the SREBP-1 gene encodes 2 proteins, SREBP-1a
and SREBP-1c/ADD1 (ADD1 is adipocyte differentiation-1) as a
consequence of alternative exon usage. 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 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: LXR and LXR. 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 3 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 2
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 8 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 regulated protein (Insig, 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 26, 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 isoforms of Insig identified as
Insig-1 and Insig-2. Insig-1 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. Insig-1 expression is highest in
human liver while Insig-2 expression is ubiquitous. The Insig
proteins bind to oxysterols which in turn affects their
interactions with SCAP. Human Insig-1 is composed of 277 amino
acids and Insig-2 contains 225. 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 PPAR, 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 2 distinct enzymes. The regulated
cleavage occurs in the lumenal loop between the 2 transmembrane
domains. This cleavage is catalyzed by site-1 protease, S1P. 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. 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.
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.
back to the top
Treatment of HypercholesterolemiaReductions 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.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: 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). For
more detailed information on the normal biological function of
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 -hydroxybutyrate or
nicotinic acid binding to GPR109A on adipocytes or macrophages.
During periods of fasting, hepatic ketone synthesis increases and
the released -butyrate bindes to GPR109A 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 GPR109A 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
GPR109A receptor on macrophages is also activated by nicotinic acid
but this effect contributes to the undesired side-effets of
nicotinic acid therapy. Within macrophages, GPR109A activation
results in increased activation of PLA2 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-
(PPAR) class of proteins that are classified as nuclear receptor
co-activators. The naturally occurring ligands for PPAR are
leukotriene B4 (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 PPAR in liver and muscle. In the liver this leads to
increased -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
520% 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
(1937%) 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.back to the top
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Michael W King, PhD | 19962013 themedicalbiochemistrypage.org,
LLC | info @ themedicalbiochemistrypage.org
Last modified: January 1, 2014