Introduction to Cholesterol Metabolism
Cholesterol is an extremely important biological molecule that has roles in membrane structure as well as being a precursor for the synthesis of the steroid hormones 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
Biosynthesis of Cholesterol
Slightly less than half of the cholesterol in the body derives from biosynthesis de novo. Biosynthesis in the liver accounts for approximately 10%, and in the intestines approximately 15%, of the amount produced each day. 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 derived from cytoplasmic oxidation of ethanol by acetyl-CoA synthetase. 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.
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 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 begins with the transport of acetyl-CoA ffrom 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 the same as those in the mitochondria. Two moles of acetyl-CoA are condensed in a reversal of the thiolase reaction, forming acetoacetyl-CoA. 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.
Mevalonate is then activated by three successive phosphorylations, yielding 5-pyrophosphomevalonate. In addition to activating mevalonate, the phosphorylations maintain its solubility, since otherwise it is insoluble in water. 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 (squalene synthase also is tightly associated with the endoplasmic reticulum). 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.
Regulating Cholesterol Synthesis
Normal healthy adults synthesize cholesterol at a rate of approximately 1g/day and consume approximately 0.3g/day. A relatively constant level of cholesterol in the body (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 (see below). 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, ACAT
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 (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, (used to be termed HMGR kinase), 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 HMG-CoA reductase 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.
Proteolytic Regulation of HMG-CoA Reductase
The stability of HMGR is regulated as the rate of flux through the mevalonate synthesis pathway changes. When the flux is high the rate of HMGR degradation is also high. When the flux is low, degradation of HMGR decreases. This phenomenon can easily be observed in the presence of the statin drugs as discussed below.
HMGR is localized to the ER and like SREBP (see below) contains a sterol-sensing domain, SSD. When sterol levels increase in cells there is a concomitant increase in the rate of HMGR degradation. The degradation of HMGR occurs within the proteosome, a multiprotein complex dedicated to protein degradation. The primary signal directing proteins to the proteosome is ubiquitination. Ubiquitin is a 7.6kDa protein that is covalently attached to proteins targeted for degradation by ubiquitin ligases. These enzymes attach multiple copies of ubiquitin allowing for recognition by the proteosome. HMGR has been shown to be ubiquitinated prior to its degradation. The primary sterol regulating HMGR degradation is cholesterol itself. As the levels of free cholesterol increase in cells, the rate of HMGR degradation increases.
The Utilization of Cholesterol
Cholesterol is transported in the plasma predominantly as cholesteryl esters associated with lipoproteins. Dietary cholesterol is transported from the small intestine to the liver within chylomicrons. Cholesterol synthesized by the liver, as well as any dietary cholesterol in the liver that exceeds hepatic needs, is transported in the serum within LDLs. The liver synthesizes VLDLs and these are converted to LDLs through the action of endothelial cell-associated lipoprotein lipase. Cholesterol found in plasma membranes can be extracted by HDLs and esterified by the HDL-associated enzyme 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.
Regulation of Cellular Sterol Content
The continual alteration of the intracellular sterol content occurs through the regulation of key sterol synthetic enzymes as well as by altering the levels of cell-surface LDL receptors. As cells need more sterol they will induce their synthesis and uptake, conversely when the need declines synthesis and uptake are decreased. Regulation of these events is brought about primarily by sterol-regulated transcription of key rate limiting enzymes and by the regulated degradation of HMGR. Activation of transcriptional control occurs through the regulated cleavage of the membrane-bound transcription factor sterol regulated element binding protein, SREBP. As discussed above, degradation of HMGR is controlled by the ubiquitin-mediated pathway for proteolysis.
Sterol control of transcription affects more than 30 genes involved in the biosynthesis of cholesterol, triacylglycerols, phospholipids and fatty acids. Transcriptional control requires the presence of an octamer sequence in the gene termed the sterol regulatory element, SRE-1. It has been shown that SREBP is the transcription factor that binds to SRE-1 elements. 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. SREBP-2 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. SREBP-1c/ADD1 controls the expression of genes involved in fatty acid synthesis.
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 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). 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.
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.
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 theNiemann-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.
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.
Treatment of Hypercholesterolemia
Drug treatment to lower plasma lipoproteins and/or cholesterol is primarily aimed at reducing the risk of athersclerosis 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 Aspirin 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 are exerted via the actions of the lipoxin family of anti-inflammatory lipids.
Additional anti-inflammatory actions of the statins results 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. Thecytokines 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). 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.
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 theperoxisome proliferator-activated receptor-α (PPAR-α) class of proteins that are classified as nuclear receptor co-activators. The naturally occurring ligands for PPAR-α 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-α 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.
Bile Acids Synthesis and Utilization
The end products of cholesterol utilization are the bile acids. Indeed, the synthesis of the bile acids is the major pathway of cholesterol catabolism in mammals. Although several of the enzymes involved in bile acid synthesis are active in many cell types, the liver is the only organ where their complete biosynthesis can occur. Synthesis of bile acids is one of the predominant mechanisms for the excretion of excess cholesterol. However, the excretion of cholesterol in the form of bile acids is insufficient to compensate for an excess dietary intake of cholesterol. Although bile acid synthesis constitutes the route of catabolism of cholesterol, these compounds are also important in the solubilization of dietary cholesterol, lipids, and essential nutrients thus promoting their delivery to the liver. Synthesis of a full complement of bile acids requires 17 individual enzymes and occurs in multiple intracellular compartments that include the cytosol, endoplasmic reticulum (ER), mitochondria, and peroxisomes. The genes encoding several of the enzymes of bile acid synthesis are under tight regulatory control to ensure that the necessary level of bile acid production is coordinated to changing metabolic conditions. Given the fact that many bile acid metabolites are cytotoxic it is understandable why their synthesis needs to be tightly controlled. Several inborn errors in metabolism are due to defects in genes of bile acid synthesis and are associated with liver failure in early childhood to progressive neuropathies in adults.
The major pathway for the synthesis of the bile acids is initiated via hydroxylation of cholesterol at the 7 position via the action of cholesterol 7α-hydroxylase (CYP7A1) which is an ER localized enzyme. CYP7A1 is a member of the cytochrome P450 family of metabolic enzymes. This pathway is depicted in highly abbreviated fashion in the Figure below. The pathway initiated by CYP7A1 is referred to as the "classic" or "neutral" pathway of bile acid synthesis. There is an alternative pathway that involves hydroxylation of cholesterol at the 27 position by the mitochondrial enzyme sterol 27-hydroxylase (CYP27A1). This alternative pathway is referred to as the "acidic" pathway of bile acid synthesis. Although in rodents the acidic pathway can account for up to 25% of total bile acid synthesis, in humans it accounts for no more than 6%. The bile acid intermediates generated via the action of CYP27A1 are subsequently hydroxylated on the 7 position by oxysterol 7α-hydroxylase (CYP7B1). Although the acidic pathway is not a major route for human bile acid synthesis it is an important one as demonstrated by the phenotype presenting in a newborn harboring a mutation in the CYP7B1 gene. This infant presented with severe cholestasis (blockage in bile flow from liver) with cirrhosis and liver dysfunction.
The hydroxyl group on cholesterol at the 3 position is in the β-orientation and must be epimerized to the α-orientation during the synthesis of the bile acids. This epimerization is initiated by conversion of the 3β-hydroxyl to a 3-oxo group catalyzed by 3β-hydroxy-Δ5-C27-steroid oxidoreductase (HSD3B7). That this reaction is critical for bile acid synthesis and function is demonstrated in children harboring mutations in the HSD3B7 gene. These children develop progressive liver disease that is characterized by cholestatic jaundice.
Following the action of HSD3B7 the bile acid intermediates can proceed via two pathways whose end products are chenodeoxycholic acid (CDCA) and cholic acid (CA). The distribution of these two bile acids is determined by the activity of sterol 12α-hydroxylase (CYP8B1). The intermediates of the HSD3B7 reaction that are acted on by CYP8B1 become CA and those that escape the action of the enzyme will become CDCA. Therefore, the activity of the CYP8B1 gene will determine the ratio of CA to CDCA. As discussed below the CYP8B1 gene is subject to regulation by bile acids themselves via their ability to regulate the action of the nuclear receptor FXR.
Synthesis of the 2 primary bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA). The reaction catalyzed by the 7α-hydroxylase (CYP7A1) is the rate limiting step in bile acid synthesis. Expression of CYP7A1 occurs only in the liver. Conversion of 7α-hydroxycholesterol to the bile acids requires several steps not shown in detail in this image. Only the relevant co-factors needed for the synthesis steps are shown. Sterol 12α-hydroxylase (CYB8B1) controls the synthesis of cholic acid and as such is under tight transcriptional control (see text).
The most abundant bile acids in human bile are chenodeoxycholic acid (45%) and cholic acid (31%). These are referred to as the primary bile acids. Before the primary bile acids are secreted into the canalicular lumen they are conjugated via an amide bond at the terminal carboxyl group with either of the amino acids glycine or taurine. These conjugation reactions yield glycoconjugates and tauroconjugates, respectively. This conjugation process increases the amphipathic nature of the bile acids making them more easily secretable as well as less cytotoxic. The conjugated bile acids are the major solutes in human bile.
Structure of the conjugated cholic acids
Following secretion by the liver the bile acids enter the bile canaliculi which join with the bile ductules which then form the bile ducts. Bile acids are carried from the liver through these ducts to the gallbladder, where they are stored for future use. In the gallbladder bile acids are concentrated up to 1000 fold. Following stimulation by food intake the gallbladder releases the bile into the duodenum, via the action of the gut hormone cholecystokinin (CCK), where they aid in the emulsification of dietary lipids.
Within the intestines the primary bile acids are acted upon by bacteria and undergo a deconjugation process that removes the glycine and taurine residues. The deconjugated bile acids are either excreted (only a small percentage) or reabsorbed by the gut and returned to the liver. Anaerobic bacteria present in the colon modify the primary bile acids converting them to the secondary bile acids, identified as deoxycholate (from cholate) and lithocholate (from chenodeoxycholate). Both primary and secondary bile acids are reabsorbed by the intestines and delivered back to the liver via the portal circulation. Indeed, as much as 95% of total bile acid synthesized by the liver is absorbed by the distal ileum and returned to the liver. This process of secretion from the liver to the gallbladder, to the intestines and finally reabsorption is termed the enterohepatic circulation.
Regulation of Bile Acid Homeostasis
Bile acids, in particular chenodeoxycholic acid (CDCA) and cholic acid (CA), can regulate the expression of genes involved in their synthesis, thereby, creating a feed-back loop. The elucidation of this regulatory pathway came about as a consequence of the isolation of a class of receptors called the farnesoid X receptors, FXRs. The FXRs belong to the superfamily of nuclear receptors that includes the steroid/thyroid hormone receptor family as well as theliver X receptors (LXRs), retinoid X receptors (RXRs), and the peroxisome proliferator-activated receptors (PPARs).
There are two genes encoding FXRs identified as FXRα and FXRβ. In humans at least four FXR isoforms have been identified as being derived from the FXRα gene as a result of activation from different promoters and the use of alternative splicing; FXRα1, FXRα2, FXRα3, and FXRα4. The FXR gene is also known as the NR1H4 gene (for nuclear receptor subfamily 1, group H, member 4). The FXR genes are expressed at highest levels in the intestine and liver.
Like all receptors of this superfamily, ligand binds the receptor in the cytoplasm and then the complex migrates to the nucleus and forms a heterodimer with other members of the family. FXR forms a heterodimer with members of the RXR family. Following heterodimer formation the complex binds to specific sequences in target genes called FXR response elements (FXREs) resulting in regulated expression. One major target of FXR is the small heterodimer partner (SHP) gene. Activation of SHP expression by FXR results in inhibition of transcription of SHP target genes. Of significance to bile acid synthesis, SHP represses the expression of the cholesterol 7α-hydroxylase gene (CYP7A1). CYP7A1 is the rate-limiting enzyme in the synthesis of bile acids from cholesterol via the classic pathway.
In the Ayurvedic tradition of medicine, any resin that is collected by tapping the trunk of a tree is called guggul (or guggal). The cholesterol lowering action of the guggul from the Mukul myrrh tree (Commiphora mukul) of India
The expression of other genes involved in bile acid synthesis is also regulated by FXR action. The action of FXR can either be to induce or repress the expression of these genes. Genes that are repressed in addition to CYP7A1 include SREBP-1c, sterol 12α-hydroxylase (gene symbol = CYP8B1), and solute carrier family 10 (sodium/bile acid cotransporter family), member 1 (gene symbol = SLC10A1). This latter gene is identified as the Na+-taurocholate cotransporting polypeptide (NTCP). NTCP is involved in hepatic uptake of bile acids through the sinusoidal/basolateral membrane. Thus bile acid-mediated repression of NTCP gene expression would reduce uptake of bile acids and protect the liver from the toxic effects of excess bile acid accumulation. Bile acids repress the transcription of another bile acid transporter that is expressed in the sinusoidal/basolateral membrane. This transporter is Na+-independent and is called the organic anion transporting polypeptide 1B1 (OATP1B1, gene symbol = SLCO1B1). OATP1B1 was formerly identified as OATP-C. The effect of bile acids on OATP1B1 expression is indirect and involves SHP-mediated reduction in HNF-4α activity which in turn reduces the expression of another liver-enriched transcription factor HNF-1α which is the major activator of the OATP1B1 gene.
Genes that, in addition to SHP, are induced by FXR include liver bile salt export pump (BSEP), multidrug resistance protein 3 (MDR3), and multidrug resistance associated protein 2 (MRP2). The latter two genes are involved in export of organic compounds and were identified based upon their ability to transport drugs out of cells thereby, allowing the cells to resist the intended effects of the administered drug. The normal function of MDR3, which is a member of the ATP-binding cassette (ABC) family of transporters (MDR3 is also identified as ABCB4), is the translocation of phospholipids through the canalicular membrane of hepatocytes. Thus, it is inferred that the bile acid-mediated increase in MDR3 expression is necessary to allow hepatocytes to respond to bile acid toxicity via the formation of cholesterol, phospholipid, and bile acid containing micelles. BSEP is also a member of the ABC family of transporters (BSEP is also identified as ABCB11) and it is involved in the process of exporting bile acids out of hepatocytes thus reducing their toxicity to these cells. Although guggulsterones antagonize the actions of FXR, which would lead to a reduction in bile acid export, these lipids have been shown to activate the expression of BSEP through an FXR-independent mechanism. This latter effect likely explains the cholesterol lowering benefits attributed to these compounds.
Of major clinical significance is that many of the FXR target genes have been implicated in several inherited cholestatic liver disorders. Mutations in BSEP and MDR3 are associated with familial intrahepatic cholestasis type 2 and 3, respectively. Mutations in MRP2 are associated with Dubin-Johnson syndrome, a form of inherited hyperbilirubinemia.
Bile Acids as Metabolic Regulators
Bile acids were originally identified as being involved in four primary physiologically significant functions:
1. their synthesis and subsequent excretion in the feces represent the only significant mechanism for the elimination of excess cholesterol.
2. bile acids and phospholipids solubilize cholesterol in the bile, thereby preventing the precipitation of cholesterol in the gallbladder.
3. they facilitate the digestion of dietary triacylglycerols by acting as emulsifying agents that render fats accessible to pancreatic lipases.
4. they facilitate the intestinal absorption of fat-soluble vitamins.
However, over the past several years new insights into the biological activities of the bile acids have been elucidated. Recent findings have demonstrated that bile acids are involved in the control of their own metabolism and transport via the enterohepatic circulation, regulate lipid metabolism, regulate glucose metabolism, control signaling events in liver regeneration, and the regulation of overall energy expenditure.
Following the isolation and characterization of the farnesoid X receptors (FXRs), for which the bile acids are physiological ligands, the functions of bile acids in the regulation of lipid and glucose homeostasis has begun to emerge. As indicated above, the binding of bile acids to FXRs results in the attenuated expression of several genes involved in overall bile acid homeostasis. However, genes involved in bile acid metabolism are not the only ones that are regulated by FXR action as a consequence of binding bile acid. In the liver, FXR action is known to regulate the expression of genes involved in lipid metabolism (e.g. SREBP-1c), lipoprotein metabolism (e.g. apoC-II), glucose metabolism (e.g. PEPCK), and hepatoprotection (e.g. CYP3A4, which was originally identified as nifedipine oxidase; nifedipine being a member of the calcium channel blocker drugs).
In addition to their roles in lipid emulsification in the intestine and activating FXR, the bile acids participate in various signal transduction processes via activation of the c-JUN N-terminal kinase (JNK) as well as the mitogen-activated protein kinase (MAPK) pathways. Other members of the nuclear receptor family that are activated by bile acids are the pregnane X receptor (PXR), the constitutive androstane receptor (CAR), and the vitamin D receptor (VDR). An additional receptor activated in response to bile acids that may have implications for control of obesity is the transmembrane G-protein coupled bile acid receptor 1 (originally identified as TGR5). Activation of TGR5 in brown adipose tissue results in activation of thermogenin (uncoupling protein 1, UCP1) leading to enhanced energy expenditure.
Inborn Errors in Bile Acids Synthesis
Metabolic disorders associated with bile acid synthesis and metabolism are broadly classified as primary or secondary disorders. Primary disorders involve inherited deficiencies in enzymes responsible for catalyzing key reactions in the synthesis of cholic and chenodeoxycholic acids. Bile acid disorders classified as secondary refer to metabolic defects that impact primary bile acid synthesis but that are not due to defects in the enzymes responsible for synthesis. Secondary disorders of bile acid metabolism include peroxisomal disorders such as Zellweger syndrome and related peroxisomal biogenesis disorders and Smith-Lemli-Opitz syndrome which results from a deficiency of 7-dehydrocholesterol reductase (DHCR7). Shown in the Table below are six of the known primary disorders of bile acid metabolism.
Affected Enzyme | Gene Symbol | Phenotype / Comments |
cholesterol 7α-hydroxylase | CYP7A1 | no liver dysfunction, clinical phenotype manifests with markedly elevated total cholesterol as well as LDL, premature gallstones, premature coronary and peripheral vascular disease, elevated serum cholesterol is not responsive to statin drug therapy |
sterol 27-hydroxylase | CYP27A1 | progressive neurological dysfunction, neonatal cholestasis, bilateral cataracts, chronic diarrhea |
oxysterol 7α-hydroxylase | CYP7B1 | a single case has been reported, a 10-week-old boy presented with severe progressive cholestasis, hepatosplenomegaly, cirrhosis, and liver failure, serum ALT and AST were markedly elevated |
3β-hydroxy-Δ5-C27-steroid oxidoreductase | HSD3B7 | most commonly reported defect in bile acid synthesis, heterogeneous clinical presentation that includes progressive jaundice, hepatomegaly, puritis, malabsorption with resultant steatorrhea (fatty diarrhea), fat soluble vitamin deficiency, rickets |
Δ4-3-oxosteroid 5β-reductase | AKR1C4 | similar clinical manifestation to HSD3B7 deficiency although with earlier presentation afflicted infants will have a more severe liver disease with rapid progression to cirrhosis and death if no clinical intervention is undertaken, liver function tests will show marked elevation in AST and ALT, serum tests show elevated conjugated bilirubin, coagulopathy will also be evident |
2-methylacyl-CoA racemase | AMACR | first reported in 3 adults who presented with a sensory motor neuropathy, also found in a 10-week-old infant who had severe fat-soluble vitamin deficiencies, hematochezia (passage of bright red stool), and mild cholestatic liver disease |
Oxidorecuctases can be either oxidases or dehydrogenases. Oxidases are generally involved when molecular oxygen functions as an acceptor of hydrogen or electrons. However, dehydrogenases work by oxidizing a substrate through transferring hydrogen to an acceptor that is either NAD/NADP or a flavin enzyme. oxidoreductase introduction
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