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, fat soluble vitamins, and other 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.
Bile salts are secreted from hepatocytes, into the bile canaliculi, via the action of the bile salt export protein (BSEP; ATP-binding cassette B11, ABCB11). Transport of phospholipids into the canaliculi requires the transporter ABCB4. ABCB4 is also known as multi-drug resistance protein 3 (MDR3, a member of the P-glycoprotein family of transporters). Some free cholesterol is also transported out of hepatocytes into the canaliculi via the action of the obligate heterodimeric transporter ABCG5/ABCG8. The transport of cholesterol via this complex also requires ABCB4. The ABCB4 requirement is consistent with the known actions of phospholipids in the bile canaliculi functioning as a sink to accept cholesterol tranported out by ABCG5/ABCG8. Each of these hepatic lipid transporters is critical for normal hepato-biliary function since mutations in any of the genes encoding the transporters have been shown to be associated with cholestatic liver diseases. These transport defects result in the accumulation of toxic levels of bile salts within the hepatocytes resulting in liver failure.
The mixture of bile salts, phospholipids and cholesterol is then transported, via the canaliculi, into the gall bladder, where they are concentrated to form bile. The composition of bile is 85% water, 67% bile salts, 22% phospholipids, and 4% cholesterol. In addition, bile contains electrolytes, minerals, minor levels of proteins, plus bilirubin and biliverdin pigments. The bilirubin and biliverdin are what impart the yellow-green or even orange hue to bile. The primary role of bile salt in the bile contained in the gall bladder is to solubilize cholesterol, thereby preventing cholesterol crystallization and the formation of cholesterol calculi (gallstones).
Following the consumption of lipid in the diet, enteroendocrine I cells in the duodenum secrete the hormone cholecystokinin (CCK) into the circulation. The release of CCK, and its subsequent binding to receptors on the gall bladder, promotes contraction of smooth muscle cells of the gall bladder and relaxation of the sphincter of Oddi, resulting in the pulsatile secretion of bile into the duodenum. Within the lumen of the duodenum the bile salt-containing mixed micelles facilitate absorption of the fat-soluble vitamins A, D, K, and E and the digestion of dietary lipids by pancreatic enzymes. Although the gall bladder stores bile, as an organ it is not essential since patients who have undergone a cholecystectomy (gall bladder removal), are still able to absorb lipids from the diet as a result of direct secretion of bile into the duodenum.
Once bile salts are secreted into the duodenum and carry out their emulsification role, around 95% are reabsorbed into the distal ileum. Bile salt reabsorption occurs via the apical sodium-dependent bile transporter (ASBT) present in the brush border membrane of the enterocyte. Ileal bile acid-binding protein (IBABP; also known as fatty acid-binding protein subclass 6: FABP6) is thought to be involved in the transport of bile salts across the enterocyte cytosol to the basolateral membrane. Once bile salts reach the basolateral membrane they are transported (effluxed) into the blood by the heterodimeric transporter OSTα/OSTβ (organic solute transporters). A small percentage of the bile salts are not reabsorbed and undergo deconjugation by intestinal microbiota before either being absorbed or converted into secondary bile acids. Anaerobic bacteria present in the colon modify the primary bile acids converting them to the secondary bile acids, identified as deoxycholate and ursodeoxycholate (DCA and UDCA, from cholate) and lithocholate (LCA, from chenodeoxycholate). These secondary bile acids are either passively absorbed from the colon or they are excreted in the feces. The absorbed primary and secondary bile acids and salts are transported back to the liver where most, but not all, are actively transported into hepatocytes by sodium sodium (Na+)-taurocholate cotransporting polypeptide (NTCP/SLC10A1) and organic anion transporters (OATP) such as OAT1B2) that mediate the uptake of bile salts and bile acids, respectively. Once in the liver the bile acids are reconjugated and then re-secreted together with newly synthesized bile salts. This overall process constitutes one cycle of what is called the enterohepatic circulation. When LCA is returned to the liver it undergoes a sulfation reaction and is subsequently excreted in the feces. The bile acid pool contains about 2–4 gm of bile acids and this pool is recycled via the enterohepatic circulation on the order of six to ten times each day. Of the total bile salt pool, around 0.2–0.6 gm are excreted in the feces each day. This lost fraction of bile salts is replenished via de novo hepatic bile acid synthesis from cholesterol.
Structure of a liver lobule. Lobules in the liver represent histologically and functionally distinct domains within the liver. They are not to be confused with the anatomical lobes of the liver which are defined as the right and left lobes and the median and quadrate lobes. Lobules are histologically defined as classical, portal, and acinus lobules. Lobules contain hepatocytes, and are vascularized by the hepatic portal vein, the hepatic artery, and the central vein. In addition, the bile cannaliculi run through the lobules allowing hepatic products such as bile acids to be delivered to the bile ducts and ultimately to the gallbladder. Kuppfer cells are liver resident macrophages.back to the top
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 the liver 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 is that a lipid component of this extract called guggulsterone (also called guggul lipid) is an antagonist of FXR. However, in addition to its effects on FXR function, guggulsterone has been shown to activate the pregnane X receptor (PXR) which is another member of the nuclear receptor superfamily. PXR is a recognized receptor for lithocholic acid and other bile acid precursors. PXR activation leads to repression of bile acid synthesis due to its physical association with hepatocyte nuclear factor 4α (HNF-4α) causing this transcription factor to no longer be able to associate with the transcriptional co-activator PGC-1α (PPARγ co-activator 1α) which ultimately leads to loss of transcription factor activation of CYP7A1.
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.back to the top
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 and also known as GPR131). Activation of TGR5 in brown adipose tissue results in activation of uncoupling protein 1, UCP1 (thermogenin) leading to enhanced energy expenditure.back to the top
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 was reported in 1998 of a 10-week-old boy presenting with severe progressive cholestasis, hepatosplenomegaly, cirrhosis, and liver failure, serum ALT and AST were markedly elevated; recently several individuals suffering from autosomal recessive hereditary spastic paraplegia 5A (SPG5A) have been shown to harbor mutations in the CYP7B1 gene although the number of cases only represents around 1% of all SPG cases|
|3β-hydroxy-Δ5-C27-steroid oxidoreductase||HSD3B7||most commonly reported defect in bile acid synthesis, heterogeneous clinical presentation that includes progressive jaundice, hepatomegaly, pruritis, 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|