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.
Slightly less than half of the cholesterol in the body derives from biosynthesis de novo. Biosynthesis in the liver accounts for approximately 10%, and in the intestines approximately 15%, of the amount produced each day. The cholesterol biosynthesis pathway involves enzymes that are in the cytoplasm, microsomes (ER), and peroxisomes. Synthesis of cholesterol, like that of most biological lipids, begins from the two-carbon acetate group of acetyl-CoA.
The acetyl-CoA utilized for cholesterol biosynthesis is derived from an oxidation reaction (e.g., fatty acids or pyruvate) in the mitochondria and is transported to the cytoplasm by the same process as that described for fatty acid synthesis (see the Figure below). Acetyl-CoA can also be synthesized from cytosolic acetate derived from cytoplasmic oxidation of ethanol which is initiated by cytoplasmic alcohol dehydrogenase (ADH). All the reduction reactions of cholesterol biosynthesis use NADPH as a cofactor. The isoprenoid intermediates of cholesterol biosynthesis can be diverted to other synthesis reactions, such as those for dolichol (used in the synthesis of N-linked glycoproteins, coenzyme Q (of the oxidative phosphorylation pathway) or the side chain of heme-a. Additionally, these intermediates are used in the lipid modification of some proteins.
Pathway for the movement of acetyl-CoA units from within the mitochondrion to the cytoplasm. Note that the cytoplasmic malic enzyme catalyzed reaction generates NADPH which can be used for reductive biosynthetic reactions such as those of fatty acid and cholesterol synthesis. SLC25A1 is the citrate transporter (also called the dicarboxylic acid transporter). Transport of pyruvate across the plasma membrane is catalyzed by the SLC16A1 protein (also called the monocarboxylic acid transporter 1, MCT1) and transport across the outer mitochondrial membrane involves a voltage-dependent porin transporter. Pyruvate transport across the inner mitochondrial membrane requires a heterotetrameric transport complex (mitochondrial pyruvate carrier) consisting of the MPC1 gene and MPC2 gene encoded proteins.
The process of cholesterol synthesis can be considered to be composed of five major steps:
1. Acetyl-CoAs are converted to 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA)
2. HMG-CoA is converted to mevalonate
3. Mevalonate is converted to the isoprene based molecule, isopentenyl pyrophosphate (IPP), with the concomitant loss of CO2
4. IPP is converted to squalene
5. Squalene is converted to cholesterol.
Pathway of cholesterol biosynthesis. Synthesis of cholesterol begins with the transport of acetyl-CoA from within the mitochondria to the cytosol. The rate limiting step in cholesterol synthesis occurs at the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reducatase, HMGR, catalyzed step. The phosphorylation reactions are required to solubilize the isoprenoid intermediates in the pathway. Intermediates in the pathway are used for the synthesis of prenylated proteins, dolichol, coenzyme Q and the side chain of heme a. The abbreviation "PP" (e.g. isopentenyl-PP) stands for pyrophosphate. ACAT2: acetyl-CoA acetyltransferase 2. HMGCS1: HMG-CoA synthase 1 (cytosolic). HMGCR: HMG-CoA reductase. MVK: mevalonate kinase. PMVK: phosphomevalonate kinase. MVD: diphosphomevalonate decarboxylase. IDI1/IDI2: isopentenyl-diphosphate delta isomerase 1 and 2. FDPS: farnesyl diphosphate synthase. GGPS1: geranylgeranyl diphosphate synthase 1. FDFT1: farnesyl-diphosphate farnesyltransferase 1 (more commonly called squalene synthase). SQLE: squalene epoxidase (also called squalene monooxygenase). LSS: lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase). DHCR7: 7-dehydrocholesterol reductase.
Acetyl-CoA units are converted to mevalonate by a series of reactions that begins with the formation of HMG-CoA. Unlike the HMG-CoA formed during ketone body synthesis in the mitochondria, this form is synthesized in the cytoplasm. However, the pathway and the necessary enzymes are similar to those in the mitochondria. Two moles of acetyl-CoA are condensed in a reversal of the thiolase reaction, forming acetoacetyl-CoA. The cytoplasmic thiolase enzyme involved in cholesterol biosynthesis is acetoacetyl-CoA thiolase (acetyl-CoA acetyltransferase 2) encoded by the ACAT2 gene. Although the bulk of acetoacetyl-CoA is derived via this process, it is possible for some acetoacetate, generated during ketogenesis, to diffuse out of the mitochondria and be converted to acetoacetyl-CoA in the cytosol via the action of acetoacetyl-CoA synthetase (AACS). Acetoacetyl-CoA and a third mole of acetyl-CoA are converted to HMG-CoA by the action of the cytosolic version of HMG-CoA synthase encoded by the HMGCS1 gene. The HMGCS1 gene is located on chromosome 5p14–p13 and is composed of 12 exons that generate two alternatively spliced mRNAs, both of which encode the same 520 amino acid protein.
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–q14 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.
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 14 exons that encode a 400 amino acid protein. Isopentenyl pyrophosphate is in equilibrium with its isomer, dimethylallyl pyrophosphate (DMPP) via the action of isopentenyl-diphosphate delta isomerase (also called isopentenylpyrophosphate isomerase). Humans express two isopentenyl-diphosphate delta isomerase genes, IDI1 and IDI2. The IDI1 gene is located on chromosome 10p15.3 and is composed of 7 exons that encode a 284 amino acid protein that is 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.
One molecule of IPP condenses with one molecule of DMPP to generate geranyl pyrophosphate, GPP. GPP further condenses with another IPP molecule to yield farnesyl pyrophosphate, FPP. Synthesis of both GPP and FPP is catalyzed by the enzyme, farnesyl diphosphate synthase. Farnesyl diphosphate synthase is derived from the FDPS gene which is located on chromosome1q22 and is composed of 11 exons that generate five alternatively spliced mRNAs that, together, encode three different isoforms of the enzyme.
The synthesis of squalene, from FPP, represents the first cholesterol-specific step in the cholesterol synthesis pathway. This is due to the fact that, as depicted in the pathway Figure above, several intermediates in the pathway can be diverted to the production of other biologically relevant molecules. The synthesis of squalene is catalyzed by the NADPH-requiring enzyme, farnesyl-diphosphate farnesyltransferase 1 (commonly called squalene synthase). Farnesyl-diphosphate farnesyltransferase 1 (encoded by the FDFT1 gene) catalyzes the two-step head-to-head condensation of two molecules of FPP, yielding squalene. The FDFT1 gene is located on chromosome 8p23.1–p22 and is composed of 14 exons that generate 11 alternatively spliced mRNAs. These 11 different FDFT1-encoded mRNAs collectively synthesize five different isoforms of farnesyl-diphosphate farnesyltransferase 1.
Squalene then undergoes a two step cyclization to yield lanosterol. This first reaction in this two-step cyclization is catalyzed by the enzyme, squalene epoxidase (also called squalene monooxygenase). This enzyme uses NADPH as a cofactor to introduce molecular oxygen as an epoxide at the 2,3 position of squalene forming the intermediate, 2,3-oxidosqualene. In the second step, this epoxide intermediate is converted to lanosterol through the action of the enzyme lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase). Squalene epoxidase is derived from the SQLE gene which is located on chromosome 8q24.1 and is composed of 12 exons that encode a protein of 574 amino acids. Lanosterol synthase is derived from the LSS gene which is located on chromosome 21q22.3 and is composed of 25 exons that generate four alternatively spliced mRNAs which together generate three distinct isoforms of the enzyme.
Through a series of 19 additional reactions, lanosterol is converted to cholesterol. These 19 reaction steps are catalyzed by nine different enzymes that are localized either to the ER or to the peroxisomes. The terminal reaction in cholesterol biosynthesis is catalyzed by the enzyme 7-dehydrocholesterol reductase encoded by the DHCR7 gene. Functional DHCR7 protein is a 55.5 kDa NADPH-requiring integral membrane protein localized to the microsomal (ER) membrane. 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
Normal healthy adults synthesize cholesterol at a rate of approximately 1g/day and consume approximately 0.3g/day. A relatively constant level of cholesterol in the blood (150–200 mg/dL) is maintained primarily by controlling the level of de novo synthesis. The level of cholesterol synthesis is regulated in part by the dietary intake of cholesterol. Cholesterol from both diet and synthesis is utilized in the formation of membranes and in the synthesis of the steroid hormones and bile acids. The greatest proportion of cholesterol is used in bile acid synthesis.
The cellular supply of cholesterol is maintained at a steady level by three distinct mechanisms:
1. Regulation of HMGR activity and levels
2. Regulation of excess intracellular free cholesterol through the activity of sterol O-acyltransferases, SOAT1 and SOAT2 with SOAT2 being the predominant activity in liver. The original designation for these enzymes was ACAT for acyl-CoA: cholesterol acyltranferase. However, this conflicts with the official ACAT enzymes, ACAT1 and ACAT2 which are acetyl-CoA acetyltransferases 1 and 2. These latter two enzymes are thiolases discussed in the Lipolysis and Fatty Acid Oxidation page.
3. Regulation of plasma cholesterol levels via LDL receptor-mediated uptake and HDL-mediated reverse transport.
Regulation of HMGR activity is the primary means for controlling the level of cholesterol biosynthesis. The enzyme is controlled by four distinct mechanisms: feed-back inhibition, control of gene expression, rate of enzyme degradation and phosphorylation-dephosphorylation.
The first three control mechanisms are exerted by cholesterol itself. Cholesterol acts as a feed-back inhibitor of pre-existing HMGR as well as inducing rapid degradation of the enzyme. The latter is the result of cholesterol-induced polyubiquitination of HMGR and its degradation in the proteosome (see proteolytic degradation below). This ability of cholesterol is a consequence of the sterol sensing domain, SSD of HMGR. In addition, when cholesterol is in excess the amount of mRNA for HMGR is reduced as a result of decreased expression of the gene. The mechanism by which cholesterol (and other sterols) affect the transcription of the HMGR gene is described below under regulation of sterol content.
Regulation of HMGR through covalent modification occurs as a result of phosphorylation and dephosphorylation. The enzyme is most active in its unmodified form. Phosphorylation of the enzyme decreases its activity. HMGR is phosphorylated by AMP-activated protein kinase, AMPK (this is not the same as cAMP-dependent protein kinase, PKA). AMPK itself is activated via phosphorylation. Phosphorylation of AMPK is catalyzed by at least 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 (CaMKKβ). CaMKKβ induces phosphorylation of AMPK in response to increases in intracellular Ca2+ as a result of muscle contraction. Visit AMPK: The Master Metabolic Regulator for more detailed information on the role of AMPK in regulating metabolism.
Regulation of HMGR by covalent modification. HMGR is most active in the dephosphorylated state. Phosphorylation 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 CaMKKβ. 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
The stability of HMGR is regulated as the rate of flux through the mevalonate synthesis pathway changes. When the flux is high the rate of HMGR degradation is also high. When the flux is low, degradation of HMGR decreases. This phenomenon can easily be observed in the presence of the statin drugs as discussed below.
HMGR is localized to the ER and like SREBP (see below) contains a sterol-sensing domain, SSD. When sterol levels increase in cells there is a concomitant increase in the rate of HMGR degradation. The degradation of HMGR occurs within the proteosome, a multiprotein complex dedicated to protein degradation. The primary signal directing proteins to the proteosome is ubiquitination. Ubiquitin is a 7.6kDa protein that is covalently attached to proteins targeted for degradation by ubiquitin ligases. These enzymes attach multiple copies of ubiquitin allowing for recognition by the proteosome. HMGR has been shown to be ubiquitinated prior to its degradation. The primary sterol regulating HMGR degradation is cholesterol itself. As the levels of free cholesterol increase in cells, the rate of HMGR degradation increases.back to the top
Cholesterol is transported in the plasma predominantly as cholesteryl esters associated with lipoproteins. Dietary cholesterol is transported from the small intestine to the liver within chylomicrons. Cholesterol synthesized by the liver, as well as any dietary cholesterol in the liver that exceeds hepatic needs, is transported in the serum within LDLs. The liver synthesizes VLDLs and these are converted to LDLs through the action of endothelial cell-associated lipoprotein lipase. Cholesterol found in plasma membranes can be extracted by HDLs and esterified by the HDL-associated enzyme lecithin-cholesterol acyltransferase, LCAT. The cholesterol acquired from peripheral tissues by HDLs can then be transferred to VLDLs and LDLs via the action of cholesteryl ester transfer protein (CETP) which is associated with HDLs. Reverse cholesterol transport allows peripheral cholesterol to be returned to the liver in LDLs. Ultimately, cholesterol is excreted in the bile as free cholesterol or as bile salts following conversion to bile acids in the liver.back to the top
Cytochrome P450 enzymes are involved in a diverse array of biological processes that includes lipid, cholesterol, and steroid metabolism as well as the metabolism of xenobiotics. The now common nomenclature used to designate P450 enzymes is CYP. There are at least 57 CYP enzymes in human tissues with eight being involved in cholesterol biosynthesis and metabolism, which includes conversion of cholesterol to bile acids. CYP metabolism of cholesterol yields several oxysterols that function as biologically active molecules such as in the activation of the liver X receptors (LXRs) and SREBP (see the next section).
CYP3A4: CYP3A4 is also known as glucocorticoid-inducible P450 and nifedipine oxidase. Nifedipine is a member of the calcium channel blocker drugs used to treat hypertension. CYP3A4 is a major hepatic P450 enzyme and is responsible for the biotransformation of nearly 60% of all commercially available drugs. With respect to cholesterol metabolism, CYP3A4 catabolizes cholesterol to 4β-hydroxycholesterol. This cholesterol derivative is one of the major circulating oxysterols and is seen at elevated levels in patients treated with anti-seizure medications such as carbamazepine, phenobarbitol, and phenytoin. The nuclear receptor, pregnane X receptor (PXR), is known to be an inducer of the CYP3A4 gene.
CYP7A1: CYP7A1 is also known as cholesterol 7α-hydroxylase and is the rate limiting enzyme in the primary pathway of bile acid synthesis referred to as the classic pathway. This reaction of bile acid synthesis plays a major role in hepatic regulation of overall cholesterol balance. Deficiency in CYP7A1 manifests with markedly elevated total cholesterol as well as LDL, premature gallstones, premature coronary and peripheral vascular disease. Treatment of this disorder with members of the statin drug family do not alleviated the elevated serum cholesterol due to the defect in hepatic diversion of cholesterol into bile acids.
CYP7B1: CYP7B1 is also known as oxysterol 7α-hydroxylase and is involved in the synthesis of bile acids via the less active secondary pathway referred to as the acidic pathway. A small percentage (1%) of individuals suffering from autosomal recessive hereditary spastic paraplegia 5A (SPG5A) have been shown to harbor mutations in the CYP7B1 gene.
CYP8B1: CYP8B1 is also known as sterol 12a-hydroxylase and is involved in the conversion of 7-hydroxycholesterol (CYP7A1 product) to cholic acid which is one of two primary bile acids and is derived from the classic pathway of bile acid synthesis. The activity of CYP8B1 controls the ratio of cholic acid over chenodeoxycholic acid in the bile.
CYP27A1: CYP27A1 is also known as sterol 27-hydroxylase and is localized to the mitochondria. CYP27A1 functions with two cofactor proteins called adrenodoxin and adrenodoxin reductase to hydroxylate a variety of sterols at the 27 position. CYP27A1 is also involved in the diversion of cholesterol into bile acids via the less active secondary pathway referred to as the acidic pathway. Deficiencies in CYP27A1 result in progressive neurological dysfunction, neonatal cholestasis, bilateral cataracts, and chronic diarrhea.
CYP39A1: CYP39A1 is also known as oxysterol 7α-hydroxylase 2. This P450 enzyme was originally identified in mice in which the CYP7B1 gene had been knocked out. The preferential substrate for CYP39A1 is 24-hydroxycholesterol, which is a major product of CYP46A1, which via CYP39A1 action is diverted into bile acid synthesis.
CYP46A1: CYP46A1 is also known as cholesterol 24-hydroxylase. This enzyme is expressed primarily in neurons of the central nervous system where it plays an important role in metabolism of cholesterol in the brain. The product of CYP46A1 action if 24S-hydroxycholesterol which can readily traverse the blood-brain-barrier to enter the systemic circulation. This pathway of cholesterol metabolism in the brain is a part of the reverse cholesterol transport process and serves as a major route of cholesterol turnover in the brain. 24S-hydroxycholesterol is a known potent activator of LXR and as such serves as an activator of the expression of LXR target genes and thus, can effect regulation of overall cholesterol metabolism not only in the brain but many other tissues as well.
CYP51A1: CYP51A1 is also referred to as lanosterol-14α-demethylase. This P450 enzyme is the only one of the eight that is involved in de novo cholesterol biosynthesis and it catalyzes the removal of the 14α-methyl group from lanosterol resulting in the generation of at least two oxysterols that, in mammalian tissues, are efficiently converted into cholesterol as well as more polar sterols and steryl esters. The oxysterols derived through the action of CYP51A1 inhibit HMGR and are also known to inhibit sterol synthesis. Knock-out of the mouse CYP51A1 homolog results in a phenotype similar to that seen in the human disorder known as Antley-Bixler syndrome (ABS). ABS represents a group of heterogeneous disorders characterized by skeletal, cardiac, and urogenital abnormalities that have frequently been associated with mutations in the fibroblast growth factor receptor 2 (FGFR2) gene.back to the top
The continual alteration of the intracellular sterol content occurs through the regulation of key sterol synthetic enzymes as well as by altering the levels of cell-surface LDL receptors. As cells need more sterol they will induce their synthesis and uptake, conversely when the need declines synthesis and uptake are decreased. Regulation of these events is brought about primarily by sterol-regulated transcription of key rate limiting enzymes and by the regulated degradation of HMGR. Activation of transcriptional control occurs through the regulated cleavage of the membrane-bound transcription factor sterol regulated element binding protein, SREBP. As discussed above, degradation of HMGR is controlled by the ubiquitin-mediated pathway for proteolysis.
Sterol control of transcription affects more than 30 genes involved in the biosynthesis of cholesterol, triacylglycerols, phospholipids and fatty acids. Transcriptional control requires the presence of an octamer sequence in the gene termed the sterol regulatory element, SRE-1. It has been shown that SREBP is the transcription factor that binds to SRE-1 elements. Humans express two distinct SREBP genes. These genes are identified as sterol regulatory element binding transcription factor 1 (SREBF1) and sterol regulatory element binding transcription factor 2 (SREBF2). In addition, mammalian SREBF1 encodes two major proteins identified as SREBP-1a and SREBP-1c/ADD1 (ADD1 is adipocyte differentiation-1) as a consequence of alternative transcriptional start sites resulting in the utilization of different first exons that are spliced to a common exon 2. The SREBF1 gene is located on chromosome 17p11.2 and is composed of 21 exons. The human SREBP-1a protein (1147 amino acids) predominates in the spleen and intestines while the SREBP-1c protein (1123 amino acids) predominates in liver, adipose tissue, and muscle. The SREBF2 gene is located on chromosome 22q13 and is composed 23 exons that encode a 1141 amino acid protein.
SREBP-1a regulates all SREBP-responsive genes in both the cholesterol and fatty acid biosynthetic pathways. SREBP-1c controls the expression of genes involved in fatty acid synthesis and is involved in the differentiation of adipocytes. SREBP-1c is also an essential transcription factor downstream of the actions of insulin at the level of carbohydrate and lipid metabolism. SREBP-2 is the predominant form of this transcription factor in the liver and it exhibits preference at controlling the expression of genes involved in cholesterol homeostasis, including all of the genes encoding the sterol biosynthetic enzymes. In addition SREBP-2 controls expression of the LDL receptor (LDLR) gene.
Regulated expression of the SREBPs is complex in that the effects of sterols are different on the SREBP-1 gene versus the SREBP-2 gene. High sterols activate expression of the SREBP-1 gene but do not exert this effect on the SREBP-2 gene. The sterol-mediated activation of the SREBP-1 gene occurs via the action of the liver X receptors (LXRs). The LXRs are members of the steroid/thyroid hormone superfamily of cytosolic ligand binding receptors that migrate to the nucleus upon ligand binding and regulate gene expression by binding to specific target sequences. There are two forms of the LXRs: LXRα and LXRβ. The LXRs form heterodimers with the retinoid X receptors (RXRs) and as such can regulate gene expression either upon binding oxysterols (e.g. 22R-hydroxycholesterol) or 9-cis-retinoic acid.
All three SREBPs are proteolytically activated and the proteolysis is controlled by the level of sterols in the cell. Full-length SREBPs have several domains and are embedded in the membrane of the endoplasmic reticulum (ER). The N-terminal domain contains a transcription factor motif of the basic helix-loop-helix (bHLH) type that is exposed to the cytoplasmic side of the ER. There are two transmembrane spanning domains followed by a large C-terminal domain also exposed to the cytosolic side. The C-terminal domain (CTD) interacts with a protein called SREBP cleavage-activating protein (SCAP). SCAP is a large protein also found in the ER membrane and contains at least eight transmembrane spans. The C-terminal portion, which extends into the cytosol, has been shown to interact with the C-terminal domain of SREBP. This C-terminal region of SCAP contains 4 motifs called WD40 repeats. The WD40 repeats are required for interaction of SCAP with SREBP. The regulation of SREBP activity is further controlled within the ER by the interaction of SCAP with insulin-induced protein-1 and -2 (Insig-1 and Insig-2: see next paragraph). When cells have sufficient sterol content SREBP and SCAP are retained in the ER via the SCAP-Insig interaction. The N-terminus of SCAP, including membrane spans 2–6, resembles HMGR which itself is subject to sterol-stimulated degradation (see above). This shared motif is called the sterol sensing domain (SSD) and as a consequence of this domain SCAP functions as the cholesterol sensor in the protein complex. When cells have sufficient levels of sterols, SCAP will bind cholesterol which promotes the interaction with Insig and the entire complex will be maintained in the ER.
There are two Insig encoding genes identified as INSIG1 and INSIG2. The INSIG1 gene is located on chromosome 7q36 and is composed of 7 exons that generate three alternatively spliced mRNAs encoding three isoforms of Insig-1. The INSIG2 gene is located on chromosome 2q14.2 and is composed of 7 exons that encode a 225 amino acid protein. The Insig-1 protein was originally isolated in experiments examining regenerating liver and was subsequently shown to be dramatically induced in fat tissue in experimental animals at the onset of diet-induced obesity. INSIG1 gene expression is highest in human liver while INSIG2 gene expression is ubiquitous. The Insig proteins bind to oxysterols which in turn affects their interactions with SCAP. The major form of human Insig-1 is a 277 amino acids protein and, as indicated, Insig-2 is a 225 amino acid protein. These two proteins share 59% amino acid identity with the greatest differences being found in the N- and C-terminal regions. Insig-2 also lacks the 50 amino acids that are found in the N-terminus of Insig-1. Both Insig proteins can cause ER retention of the SREBP/SCAP complex. The Insig proteins span the ER membrane six times. It has been shown that a critical aspartate (D) residue in Insig-1 and Insig-2, found in the cytosolic loop between membrane spans 4 and 5, is critical for interaction with SCAP as mutation of this amino acid causes loss of SCAP binding. The third and fourth transmembrane spans in both Insig proteins are required for interaction with oxysterols. The Insig-1 gene has been shown to be transcriptionally regulated by SREBP with the SRE in the Insig-1 gene residing approximately 380bp upstream of the transcriptional start site. Expression of Insig-1 has also been shown to be regulated by several members of the nuclear receptor family including PPARδ, PXR and CAR. The Insig-2 promoter is activated in response to signals downstream of insulin receptor activation. Nuclear receptors also regulate the expression of the Insig-2 gene which has been shown to contain two FXR response elements.
In addition to their role in regulating sterol-dependent gene regulation, both Insig proteins activate sterol-dependent degradation of HMGR. In the presence of the cholesterol-derived oxysterol, 24,25-dihydrolanosterol, Insig binds to the transmembrane domain of HMGR. The oxysterol-induced interaction between Insig and HMGR within the ER membrane allows Insig to recruit the ubiquitin ligase, gp78, to HMGR resulting in ubiquitination of HMGR and its resultant proteasomal degradation as described above.
When sterols are scarce, SCAP does not interact with Insig. Under these conditions the SREBP-SCAP complex migrates to the Golgi where SREBP is subjected to proteolysis. The cleavage of SREBP is carried out by two distinct enzymes. The regulated cleavage occurs in the lumenal loop between the 2 transmembrane domains. This cleavage is catalyzed by site-1 protease, S1P (also known as subtilisin/kexin-isozyme 1, SKI-1). S1P is officially called membrane-bound transcription factor peptidase, site 1, MBTPS1. The MBTPS1 gene is located on chromosome 16q24 and is composed of 23 exns that encode a 1052 amino acid preproprotein. MBTPS1 is a member of the subtilisin-like proprotein convertase 2 family of serine proteases. This family of proteases are responsible for the processing of proteins that are in the regulated or constitutive branches of the secretory pathway. The subtilisin-like proprotein convertase 2 family of enzymes are encoded by nine different genes in humans one of which is the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene whose encoded enzyme is a recent target in the treatment of hypercholesteremia (see next section). The function of SCAP is to positively stimulate S1P-mediated cleavage of SREBP.
The second cleavage, catalyzed by site-2 protease, S2P, occurs in the first transmembrane span, leading to release of active SREBP. The official name for S2P is membrane-bound trasncription factor peptidase, site 2 (MBTPS2). The MBTPS2 gene is located on the X chromosome (Xp22.12-p22.11) and is composed of 11 exons that encode a 519 amino acid protein. S2P is an intramembrane zinc metalloprotease. In order for S2P to act on SREBP, site-1 must already have been cleaved. The result of the S2P cleavage is the release of the N-terminal bHLH motif into the cytosol. The bHLH domain then migrates to the nucleus where it will dimerize and form complexes with transcriptional coactivators leading to the activation of genes containing the SRE motif. To control the level of SREBP-mediated transcription, the soluble bHLH domain is itself subject to rapid proteolysis. In addition to the cleavage-activation of SREBP transcriptional activity, S2P is involved in pathways that regulate cellular responses to endoplasmic reticulum stress, primarily the unfolded protein response, UPR.
Protease-mediated regulation of SREBP activation. Diagramatic representation of the interactions between SREBP, SCAP and Insig in the membrane of the ER when sterols are high. When sterols are low, SCAP does not interact with Insig and the SREBP-SCAP complex migrates to the Golgi where the proteases, S1P and S2P reside. bHLH = basic helix-loop-helix domain. CTD = C-terminal domain. WD = WD40 domain.
Several proteins whose functions involve sterols also contain the SSD. These include patched, an important development regulating receptor whose ligand, hedgehog, is modified by attachment of cholesterol and the Niemann-Pick disease type C1 (NPC1) protein which is involved in cholesterol transport in the secretory pathway. NPC1 is one of several genes whose activities, when disrupted, lead to severe neurological dysfunction.
Reductions in circulating cholesterol levels can have profound positive impacts on cardiovascular disease, particularly on atherosclerosis, as well as other metabolic disruptions of the vasculature. Control of dietary intake is one of the easiest and least cost intensive means to achieve reductions in cholesterol. Recent studies in laboratory rats has demonstrated an additional benefit of reductions in dietary cholesterol intake. In these animals it was observed that reductions in dietary cholesterol not only resulted in decreased serum VLDLs and LDLs, and increased HDLs but DNA synthesis was also shown to be increased in the thymus and spleen. Upon histological examination of the spleen, thymus and lymph nodes it was found that there was an increased number of immature cells and enhanced mitotic activity indicative of enhanced proliferation. These results suggest that a marked reduction in serum LDLs, induced by reduced cholesterol intake, stimulates enhanced DNA synthesis and cell proliferation.
Drug treatment to lower plasma lipoproteins and/or cholesterol is primarily aimed at reducing the risk of atherosclerosis and subsequent coronary artery disease that exists in patients with elevated circulating lipids. Drug therapy usually is considered as an option only if non-pharmacologic interventions (altered diet and exercise) have failed to lower plasma lipids.
Alirocumab (Praluent®), Evolcumab (Repatha®): These drugs are the newest type of anti-hypercholesterolemia drugs recently approved by the FDA for use in the US. Both drugs are injectible antibodies that block the function of proprotein convertase subtilisin/kexin type 9, PCSK9. PCSK9 is serine protease of the subtilisin-like proprotein convertase 2 family. A major function of PCSK9 is the endosomal degradation of the LDL receptor (LDLR), thereby reducing the recyling of the LDLR to the plasma membrane. This effect of PCSK9 leads to a reduced ability of the liver to remove IDL and LDL from the blood contributing to the potential for hypercholesterolemia. The potential for the pharmaceutical benefits of the interference in the activity PCSK9 was recognized by a confluence of several studies. Patients with a specific form of familial hypercholesterolemia not due to mutations in the LDLR gene were shown to have severe hypercholesterolemia due to mutations in the PCSK9 gene resulting in hyperactivity of the enzyme. In addition, it was found that in certain individuals with low serum LDL levels there was an association with the inheritance of nonsense mutations in the PCSK9 gene which result in loss of PCSK9 activity. Hypercholesterolemic patients taking another cholesterol-lowering drug while simultaneously utilizing either of these new PCSK9 inhibitors saw further reductions in serum LDL levels of betweeen 55% and 77%.
Atorvastatin (Lipitor®), Simvastatin (Zocor®), Lovastatin (Mevacor®): These drugs are fungal HMG-CoA reductase (HMGR) inhibitors and are members of the family of drugs referred to as the statins. The net result of treatment is an increased cellular uptake of LDLs, since the intracellular synthesis of cholesterol is inhibited and cells are therefore dependent on extracellular sources of cholesterol. However, since mevalonate (the product of the HMG-CoA reductase reaction) is required for the synthesis of other important isoprenoid compounds besides cholesterol, long-term treatments carry some risk of toxicity. A component of the natural cholesterol lowering supplement, red yeast rice, is in fact a statin-like compound.
The statins have become recognized as a class of drugs capable of more pharmacologic benefits than just lowering blood cholesterol levels via their actions on HMGR. Part of the cardiac benefit of the statins relates to their ability to regulate the production of S-nitrosylated COX-2. COX-2 is an inducible enzyme involved in the synthesis of the prostaglandins and thromboxanes as well as the lipoxins and resolvins. The latter two classes of compounds are anti-inflammatory lipids discussed in the Lipid-Derived Inflammatory Modulators page. Evidence has shown that statins activate inducible nitric oxide synthase (iNOS) leading to nitrosylation of COX-2. The S-nitrosylated COX-2 enzyme produces the lipid compound 15R-hydroxyeicosatetraenoic acid (15R-HETE) which is then converted via the action of 5-lipoxygenase (5-LOX) to the epimeric lipoxin, 15-epi-LXA4. This latter compound is the same as the aspirin-triggered lipoxin (ATL) that results from the aspirin-induced acetylation of COX-2. Therefore, part of the beneficial effects of the statins is exerted via the actions of the lipoxin family of anti-inflammatory lipids.
Additional anti-inflammatory actions of the statins result from a reduction in the prenylation of numerous pro-inflammatory modulators. Prenylation refers to the addition of the 15 carbon farnesyl group or the 20 carbon geranylgeranyl group to acceptor proteins. The isoprenoid groups are attached to cysteine residues at the carboxy terminus of proteins in a thioether linkage (C-S-C). A common consensus sequence at the C-terminus of prenylated proteins has been identified and is composed of CAAX, where C is cysteine, A is any aliphatic amino acid (except alanine) and X is the C-terminal amino acid. In addition to numerous prenylated proteins that contain the CAAX consensus, prenylation is known to occur on proteins of the RAB family of RAS-related G-proteins. There are at least 60 proteins in this family that are prenylated at either a CC or CXC element in their C-termini. The RAB family of proteins are involved in signaling pathways that control intracellular membrane trafficking. The prenylation of proteins allows them to be anchored to cell membranes. In addition to cell membrane attachment, prenylation is known to be important for protein-protein interactions. Thus, inhibition of this post-translational modification by the statins interferes with the important functions of many signaling proteins which is manifest by inhibition of inflammatory responses.
Some of the effects on immune function that have been attributed to the statins are attenuation of autoimmune disease, inhibition of T-cell proliferation, inhibition of inflammatory co-stimulatory molecule expression, decreases in leukocyte infiltration, and promotion of a shift in cytokine profiles of helper T-cell types from Th1 to Th2. Th1 cells are involved in cell-mediated immunity processes, whereas, Th2 cells are involved in humoral immunity process. The cytokines produced by Th2 cells include IL-4, IL-5, IL-10 and IL-13 and these trigger B cells to switch to IgE production and to activate eosinophils.
Nicotinic acid (Niacor® and Niaspan®): Nicotinic acid reduces the plasma levels of both VLDLs and LDLs by inhibiting hepatic VLDL secretion, as well as suppressing the flux of FFA release from adipose tissue by inhibiting lipolysis. In addition, nicotinic administration strongly increases the circulating levels of HDLs. Patient compliance with nicotinic acid administration is sometimes compromised because of the unpleasant side-effect of flushing (strong cutaneous vasodilation). Recent evidence has shown that nicotinic acid binds to and activates the G-protein coupled receptor identified as GPR109A (also called HM74A or PUMA-G). 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 β-hydroxybutyrate or nicotinic acid binding to GPR109A on adipocytes or macrophages. During periods of fasting, hepatic ketone synthesis increases and the released β-hydroxybutyrate binds 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-α (PPARα) class of proteins that are classified as nuclear receptor co-activators. The naturally occurring ligands for PPARα 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 PPARα in liver and muscle. In the liver this leads to increased β-oxidation of fatty acids, thereby decreasing the liver's secretion of triacylglycerol- and cholesterol-rich VLDLs, as well as increased clearance of chylomicron remnants, increased levels of HDLs and increased lipoprotein lipase activity which in turn promotes rapid VLDL turnover.
Cholestyramine or colestipol (resins): These compounds are nonabsorbable resins that bind bile acids which are then not reabsorbed by the liver but excreted. The drop in hepatic reabsorption of bile acids releases a feedback inhibitory mechanism that had been inhibiting bile acid synthesis. As a result, a greater amount of cholesterol is converted to bile acids to maintain a steady level in circulation. Additionally, the synthesis of LDL receptors increases to allow increased cholesterol uptake for bile acid synthesis, and the overall effect is a reduction in plasma cholesterol. This treatment is ineffective in homozygous FH patients, since they are completely deficient in LDL receptors.
Ezetimibe: This drug is sold under the trade names Zetia® or Ezetrol® and is also combined with the statin drug simvastatin and sold as Vytorin® or Inegy®. Ezetimibe functions to reduce intestinal absorption of cholesterol, thus effecting a reduction in circulating cholesterol. The drug functions by inhibiting the intestinal brush border transporter involved in absorption of cholesterol. This transporter is known as Niemann-Pick type C1-like 1 (NPC1L1). NPC1L1 is also highly expressed in human liver. The hepatic function of NPC1L1 is presumed to limit excessive biliary cholesterol loss. NPC1L1-dependent sterol uptake is regulated by cellular cholesterol content. In addition to the cholesterol lowering effects that result from inhibition of NPC1L1, its' inhibition has been shown to have beneficial effects on components of the metabolic syndrome, such as obesity, insulin resistance, and fatty liver, in addition to atherosclerosis. Ezetimibe is usually prescribed for patients who cannot tolerate a statin drug or a high dose statin regimen. There is some controversy as to the efficacy of ezetimibe at lowering serum cholesterol and reducing the production of fatty plaques on arterial walls. The combination drug of ezetimibe and simvastatin has shown efficacy equal to or slightly greater than atorvastatin (Lipitor®) alone at reducing circulating cholesterol levels.
New Approaches: Numerous epidemiological and clinical studies over the past 10 years have demonstrated a direct correlation between the circulating levels of HDL cholesterol (most often abbreviated HDL-c) and a reduction in the potential for atherosclerosis and coronary heart disease (CHD). Individuals with levels of HDL above 50mg/dL are several time less likely to experience CHD than individuals with levels below 40mg/dL. In addition, clinical studies in which apolipoprotein A-I (apoA-I), the predominant protein component of HDL-c) or reconstituted HDLs are infused into patients raises circulating HDL levels and reduces the incidence of CHD. Thus, there is precedence for therapies aimed at raising HDL levels in the treatment and prevention of atherosclerosis and CHD. Unfortunately current therapies only modestly elevate HDL levels. Both the statins and the fibrates have only been shown to increase HDL levels between 5–20% and niacin is poorly tolerated in many patients. Therefore, alternative strategies aimed at increasing HDL levels are being tested. Cholesterol ester transfer protein (CETP) is secreted primarily from the liver and plays a critical role in HDL metabolism by facilitating the exchange of cholesteryl esters (CE) from HDL for triglycerides (TG) in apoB containing lipoproteins, such as LDL and VLDL. The activity of CETP directly lowers the cholesterol levels of HDLs and enhances HDL catabolism by providing HDLs with the TG substrate of hepatic lipase. Thus, CETP plays a critical role in the regulation of circulating levels of HDL, LDL, and apoA-I. It has also been shown that in mice naturally lacking CETP most of their cholesterol is found in HDL and these mice are relatively resistant to atherosclerosis. The potential for the therapeutic use of CETP inhibitors in humans was first suggested when it was discovered in 1985 that a small population of Japanese had an inborn error in the CETP gene leading to hyperalphalipoproteinemia and very high HDL levels. To date three CETP inhibitors have been used in clinical trials. These compounds are anacetrapib, torcetrapib, and dalcetrapib. Although torcetrapib is a potent inhibitor of CETP, its' use has been discontinued due to increased negative cardiovascular events and death rates in test subjects. Treatment with dalcetrapib results in increases in HDL (19–37%) and a modest decrease (≈6%) in LDL levels. Treatment with anacetrapib results in a significant increase in both HDL (≈130%) and LDL (≈40%). Anacetrapib is currently in phase III clinical studies.back to the top