2019 Volume 67 Issue 4 Pages 333-340
Biliary lipids consist mainly of bile salts, phospholipids and cholesterol, which form mixed micelles and vesicles. Bile salts play various physiological roles but have damaging effects on cell membranes due to their detergent properties. The cytotoxicity of bile salts on hepatocytes leads to liver injuries and is largely determined by the bile salt species, the concentrations of bile salts, phospholipids and cholesterol, and the lipid composition of cell membranes. In bile, monomers and simple micelles of bile salts coexist with mixed micelles and vesicles in dynamic equilibrium, and contribute to the cytotoxicity on hepatocytes. The ATP-binding cassette (ABC) transporter family members, ABCB11, ABCB4 and ABCG5/ABCG8, mediate the biliary secretion of bile salts, phospholipids and cholesterol, respectively. Mutations in ABCB4 result in severe cholestatic diseases, and the biliary phospholipids are necessary for the attenuation of bile salt cytotoxicity. On the other hand, cholesterol reverses the cytoprotective effects of phospholipids against bile salts. In addition, phosphatidylethanolamine N-methyltransferase increases the cell resistance to bile salts by changing the phospholipid composition and structures of the apical membranes. In this review, we focus on the molecular mechanisms for the protection of hepatocytes against bile salt cytotoxicity. Further understanding of these mechanisms will help to develop new therapeutic strategies for cholestatic liver diseases.
Bile salts, phospholipids and cholesterol are major biliary lipids. Bile also contains bilirubin, proteins, mucins, electrolytes, etc. The concentrations of bile salts, phospholipids and cholesterol in bile differ between individuals and are mainly determined by ATP-binding cassette (ABC) transporters, i.e., ABCB11 for bile salts, ABCB4 for phospholipids and ABCG5/ABCG8 for cholesterol.1–6) Bile salts damage cells by affecting the integrity of cell membranes due to their potent detergent properties.7) In liver injury, an increase in bile salt cytotoxicity in bile canaliculi leads to cholestasis and hepatocellular necrosis. In normal bile, bile salts are associated with phospholipids and cholesterol in mixed micelles or vesicles, which strongly attenuates the detergent activity and cytotoxicity of bile salts.8) In this review, we describe the current understanding of how hepatocytes are protected from bile salt toxicity.
Bile salts play multiple physiological roles in induction of bile flow, cholesterol homeostasis, intestinal absorption of lipids and vitamins, digestion of proteins, glucose metabolism, detoxification, immunological signaling and antimicrobial defense.9–11) In addition, bile salts are important signaling molecules activating receptors including farnesoid X receptor, vitamin D receptor, pregnane X receptor, Takeda G protein-coupled receptor 5 (TGR5), α5β1 integrin, and sphingosine-1-phosphate receptor 2.11–13) Bile salts are efficiently reabsorbed from the intestine and undergo enterohepatic circulation.
In humans, cholate and chenodeoxycholate are primary bile salts synthesized from cholesterol in the liver, and deoxycholate and lithocholate are secondary bile salts formed by bacterial modifications of primary bile salts in the intestine9–11,14) (Fig. 1). Ursodeoxycholate is a tertiary bile salt produced via modification of a secondary bile salt, 7-ketolithocholate, by hepatocytes.9,10) In humans, cholate, deoxycholate and chenodeoxycholate are major unconjugated bile salts. In the liver, the majority of bile salt molecules are conjugated with glycine or taurine (Fig. 2) and, to a lesser extent, with glucuronate, N-acetylglucosamine or sulfate.9,10,15) The proportions of bile salts in the human gallbladder are in the order; glycochenodeoxycholate (GCDC) ≥ glycocholate (GC) > glycodeoxycholate (GDC) ≥ taurochenodeoxycholate (TCDC) ≥ taurocholate (TC) > taurodeoxycholate (TDC).16) Tauroursodeoxycholate (TUDC), glycoursodeoxycholate (GUDC), taurolithocholate (TLC) and glycolithocholate (GLC) are present in very small amounts in the human gallbladder.16)
Cholate (A) and chenodeoxycholate (B) are primary bile salts synthesized from cholesterol in hepatocytes. Deoxycholate (C) and lithocholate (D) are secondary bile salts converted from primary bile salts by intestinal bacteria. Ursodeoxycholate (E) is a tertiary bile salt produced from a secondary bile salt in hepatocytes. Cholate (A) is a trihydroxylated bile salt. Chenodeoxycholate (B), deoxycholate (C) and ursodeoxycholate (E) are dihydroxylated bile salts. Lithocholate (D) is a monohydroxylated bile salt.
Taurocholate (A) and glycocholate (B) are taurine- and glycine-conjugated forms of cholate, respectively.
In bile, the predominant phospholipid is phosphatidylcholine (PC) (>95%).15,17) Bile also contains small amounts of phosphatidylethanolamine (PE) and phosphatidylserine.15) Only trace amounts of sphingomyelin (SM) are present in bile, although the major structural phospholipids of the outer leaflet of the canalicular membranes are PC and SM.18,19) In human bile, the PC molecule contains mainly saturated acyl chain (16 : 0) at the sn-1 position and unsaturated acyl chain (18 : 2 > 18 : 1 > 20 : 4) at the sn-2 position.14) In rat bile, the SM molecule contains mainly 16 : 0 acyl chain.20)
In bile, cholesterol is unesterified and accounts for more than 97% of total sterols.17) Small amounts of cholesterol precursors and dietary phytosterols are also present in the bile.17) The water solubility of cholesterol is extremely low (3.7 × 10−8 M).21) An increase in the cholesterol concentration in bile leads to supersaturation of cholesterol, subsequent precipitation of cholesterol crystals and formation of cholesterol gallstones.14)
ABCB11, also known as bile salt export pump (BSEP) or sister of P-glycoprotein (SPGP), mediates most of the bile salt secretion from hepatocytes into the bile canalicular lumen, which is a major driving force of bile flow2,10,14) (Fig. 3). The bile salt molecules enter the canalicular space as monomers.14) Human ABCB11 has a 1321-amino acid sequence with 69% similarity to the multidrug transporter P-glycoprotein (ABCB1). The two homologous halves of the ABCB subfamily member are arranged in tandem, with each half bearing six transmembrane domains and a cytoplasmic nucleotide-binding fold.2) ABCB11 is expressed almost exclusively in the hepatocytes and localizes to the canalicular membranes and subcanalicular vesicles.22) The rank order of bile salt transport activity is TCDC > TC ≥ TUDC > GC = cholate.22)
At the hepatocyte canalicular membranes, ABCB11, ABCB4 and ABCG5/ABCG8 mediate the efflux of bile salts, phospholipids and cholesterol, respectively, using the energy of ATP hydrolysis. Bile salt molecules are secreted as monomers, and spontaneously form simple micelles above their CMCs. The phospholipid efflux activity of ABCB4 requires the secreted bile salts. The cholesterol efflux activity of ABCG5/ABCG8 requires the secreted phospholipids. Bile salts, phospholipids and cholesterol form mixed micelles. The increase in the phospholipid content in mixed micelles leads to the conversion into vesicles. The bilayer discs are intermediates in the conversion of mixed micelles to vesicles. Multilamellar vesicles are formed by the fusion of unilamellar vesicles and involved in the initiation of cholesterol crystallization.
Mutations in the gene encoding ABCB11 are responsible for progressive familial intrahepatic cholestasis type 2 (PFIC2), a fatal liver disease with greatly reduced bile flow.2) In humans, ABCB11 is the sole bile salt transporter, because almost no bile salt (<1% of normal) is present in the bile of PFIC2 patients.23) In addition, the biliary phospholipid and cholesterol concentrations are severely reduced in the PFIC2 patient.23) In patients with PFIC2, severe cholestasis, severe pruritus and hepatic inflammation occur with marked elevation of serum bile salts but with normal serum γ-glutamyl-transpeptidase activity.2,23,24) This disease rapidly progresses to cirrhosis, leading to liver failure.
On the other hand, Abcb11 knockout mice exhibit very mild cholestasis and liver steatosis.25) The bile flow in Abcb11 knockout mice is decreased by only 18% compared with that in normal mice.25) Mouse Abcb11 is the major transporter for hydrophobic bile salts, such as taurocholate, but not for hydrophilic bile salts, such as tauro-β-muricholate and tauro-ω-muricholate.25) However, Abcb1a/Abcb1b/Abcb11 triple-knockout mice show a more severe phenotype with impaired bile formation, jaundice and increased mortality, suggesting that Abcb1a and Abcb1b compensate for the lack of Abcb11.26)
ABCB4, also called multidrug resistance 3 (MDR3), is expressed on the canalicular membranes of hepatocytes and responsible for the biliary secretion of phospholipids, which is implicated in the protection of cell membranes against bile salts and in the solubilization of cholesterol4,6) (Fig. 3). Human ABCB4 is a 1279-amino acid transmembrane protein, which has 86 and 66% similarity to ABCB1 and ABCB11, respectively. We have demonstrated, using HEK293 cells stably expressing ABCB4, that the cellular phospholipid efflux mediated by ABCB4 is markedly stimulated by TC below the critical micelle concentration (CMC).27) Thus, the monomer form of TC is likely to function in supporting the ABCB4-mediated phospholipid efflux. The phospholipid efflux mediated by ABCB4 increases in the order of cholate < GC < TC.27) In the presence of TC, ABCB4 preferentially mediates the efflux of PC compared with that of PE or SM.27,28) Furthermore, we have shown that ABCB4 is predominantly distributed into nonraft membranes, but not raft membranes.28) ABCB4 has been considered to be a floppase translocating phospholipids from the inner leaflet to the outer leaflet of the cell membrane, or to be an exporter moving phospholipids directly into the extracellular space in the presence of bile salts.4,6,29–31)
In humans, the ABCB4 gene mutations lead to progressive familial intrahepatic cholestasis type 3 (PFIC3).32,33) PFIC3 is characterized by high γ-glutamyl transpeptidase and early onset of persistent cholestasis, which progresses to cirrhosis and liver failure before adulthood. Liver transplantation is the only therapy for many cases of PFIC3. The biliary concentration of phospholipids is substantially reduced in the PFIC3 patient.32) This cholestasis is attributed to the bile salt cytotoxicity leading to injuries of the bile canaliculus and biliary epithelium. ABCB4 mutations are also related to intrahepatic cholestasis of pregnancy, low phospholipid-associated cholelithiasis, primary biliary cirrhosis, and cholangiocarcinoma.4,6,24,34)
Abcb4 knockout mice show an almost complete absence of phospholipids and cholesterol in bile despite normal biliary secretion of bile salts.35) These mice develop liver disease characterized by severe necrotic damage of hepatocytes, strong portal inflammation, proliferation of bile ducts, fibrosis and gallstone formation.35,36) In normal mice, the biliary phospholipid secretion increases with increasing bile salt secretion, whereas the phospholipid secretion in Abcb4 knockout mice is negligible at all bile salt secretion rates.37)
PC is synthesized via the cytidine diphosphate-choline pathway or through the methylation of PE by PE N-methyltransferase (PEMT) in the mammalian livers.38) The PEMT-controlled pathway accounts for ca. 30% of hepatic PC production.39) Pemt knockout mice exhibit severe liver failure within 3 d and die in 4–5 d when fed a choline-deficient diet.40) However, when mice lacking both Pemt and Abcb4 are fed a choline-deficient diet, they escape liver failure and survive for more than 90 d.40) These findings have suggested that, in Pemt knockout mice fed a choline-deficient diet, the liver failure is attributed to the rapid depletion of hepatic PC via biliary secretion mediated by Abcb4 during a time when PC biosynthesis is severely curtailed, and that Pemt compensates for the lack of dietary choline. Meanwhile, the lack of Pemt leads to a reduction in the liver damage in Abcb4 knockout mice, which may be due to the decreased concentration of bile salts in the livers.41)
The P-type ATPase ATP8B1 functions as a flippase in the canalicular membranes and translocates phospholipids, PC or phosphatidylserine, from the outer to the inner leaflet.31,42) Mutations in the ATP8B1 gene cause progressive familial intrahepatic cholestasis type 1 (PFIC1) with typical cholestatic symptoms, including elevated serum bile salt, bilirubin and transaminase levels, low γ-glutamyl transpeptidase levels, and intractable pruritus.24,43,44) Groen et al. have created Abcb4/Atp8b1 double-knockout mice.31) Although the biliary phospholipid secretion in these double-knockout mice is as low as in Abcb4 knockout mice, hepatic damage in the double-knockout mice was less pronounced than that in Abcb4 knockout mice.31) The canalicular membranes of the double knockout mice are more resistant to bile salts than those of Atp8b1 knockout mice or Abcb4 knockout mice, suggesting that Atp8b1 counteracts the membrane destabilization by Abcb4.31)
Most recently, Wang et al. have generated Abcb4/Abcb11 double-knockout mice, which show no significant liver injury and have almost no gallstones, despite a lack of biliary PC.45) In the bile of these double-knockout mice, hydrophilic β-muricholate and tetrahydroxylated bile salts are increased, but hydrophobic cholate is reduced, suggesting that the hydrophilic shift in bile salt composition alleviates liver damage caused by the lack of biliary phospholipids.45)
ABCG5 and ABCG8 are expressed exclusively in the canalicular membranes of liver and intestine, and form a heterodimer1,5,14,46) (Fig. 3). Human ABCG5 and ABCG8 are present on nearly contiguous genes. The ABCG5/ABCG8 heterodimer is the main transporter for the secretion of cholesterol and plant sterols into bile. Human ABCG5 and ABCG8 are 651-amino acid and 672-amino acid transmembrane proteins, respectively, and share 46% similarity. Dog gallbladder cells double-transfected with mouse Abcg5 and Abcg8 genes show increased cholesterol efflux in the presence of bile salts, TDC, TC and TUDC, at concentrations above their CMCs, which is further enhanced by the addition of PC.47) Small has proposed a model in which ABCG5/ABCG8 lifts cholesterol partly into the canalicular lumen by ATP hydrolysis, and this exposed cholesterol is picked up by bile salt/PC mixed micelles.1)
In humans, mutations in either ABCG5 or ABCG8 result in sitosterolemia, an autosomal-recessive disorder characterized by an accumulation of plant sterols in the plasma and early coronary atherosclerosis.1,5) In contrast, a mutation of ABCG8 D19H induces higher cholesterol efflux efficacy and is strongly associated with gallstone disease.48)
Similar to the human genes, the mouse Abcg5 and Abcg8 genes are located within 400 bp of each other. Yu et al. have reported that the disruption of both Abcg5 and Abcg8 genes in mice dramatically reduces the biliary cholesterol level but does not significantly alter the biliary bile salt and phospholipid levels. The overexpression of human ABCG5/ABCG8 in mice markedly promotes the biliary cholesterol secretion, whereas Abcb4 knockout mice overexpressing human ABCG5/ABCG8 have only trace amounts of biliary cholesterol and phospholipids, suggesting that the ABCB4-mediated secretion of phospholipids is necessary for the secretion of cholesterol via ABCG5/ABCG8.49,50) Yamanashi et al. have shown that the hepatic overexpression of human Niemann–Pick C2 in mice stimulates the Abcg5/Abcg8-mediated biliary secretion of cholesterol, but not the secretion of phospholipids or bile salts, suggesting that Niemann–Pick C2 is a positive regulator of the ABCG5/ABCG8-mediated cholesterol efflux.51)
Bile salt molecules are quasi-planar amphiphiles consisting of a hydrophobic steroid nucleus with polar hydroxyl groups on one face and a flexible aliphatic side chain with a carboxy group, which is amidated with glycine or taurine (Figs. 1, 2). Cholate contains 3α-, 7α-, 12α-hydroxy groups. Chenodeoxycholate, deoxycholate or ursodeoxycholate has two hydroxy groups at 3α-, 7α-positions, 3α-, 12α-positions or 3α-, 7β-positions in the steroid nucleus, respectively. Ursodeoxycholate is a structural isomer of chenodeoxycholate. Lithocholate, a 3α-monohydroxylated bile salt, is highly hydrophobic. In addition, the conjugation with taurine or glycine increases the hydrophilicity of each bile salt. The hydrophobicities and detergent properties of bile salts are determined by the number, position and orientation of hydroxy groups in the steroid nucleus and by the conjugation of the side chain.52,53)
Above the CMC, bile salt monomers spontaneously form simple micelles. The relative balance between hydrophobic and hydrophilic planes of the steroid structure is involved in the micelle formation.9,52) The primary micelles of bile salts are formed by the interactions between the hydrophobic planes of steroid structures, and the secondary micelles are formed by the association of the primary micelles via intermolecular hydrogen bonding between the hydroxy groups.54) The CMCs increase in the order of TDC < GDC < TCDC < GCDC < TC < GC < TUDC < GUDC.55) The CMCs of taurine-conjugated bile salts are slightly lower than those of the corresponding glycine-conjugated bile salts.
Heuman has defined the bile salt monomeric hydrophobicity indices derived from the relative retention times during HPLC.53) The hydrophobicity indices, from hydrophilic to hydrophobic, are TUDC < GUDC < TC < GC < TCDC < GCDC < TDC < GDC < TLC < GLC.53) It is noteworthy that, TUDC or GUDC having two hydroxy groups in the steroid nucleus is more hydrophilic than the structural isomer, TCDC or GCDC, and than TC or GC having three hydroxy groups.
Bile salts can disrupt phospholipid bilayer membranes, leading to the formation of mixed micelles. In mixed micelles, the hydrophobic face of the bile salt molecule can interact with the phospholipid acyl chain region. This interaction prevents the contact of the acyl chain with water, whereas the hydrophilic aliphatic side chain and hydroxy face of the bile salt molecule and the phospholipid polar head region are in contact with water. At low ratios of bile salt/phospholipid, phospholipid molecules form bilayer membrane vesicles with incorporated bile salt molecules. At high bile salt/phospholipid ratios, phospholipid molecules are intercalated within bile salt molecules, and small mixed micelles are formed.15,56,57) In the vesicle-to-micelle transition model, bile salt molecules bind and penetrate into the phospholipid bilayer membranes, and subsequently the saturation of the phospholipid membranes with bile salt molecules induces the solubilization of membranes into bile salt/phospholipid mixed micelles.58) Between phase boundaries in the vesicle-to-micelle transition of egg PC and cholate mixtures, three structures, open vesicles, large bilayer sheets and long flexible cylindrical micelles, were observed by cryo-transmission electron microscopy.56)
The binding affinity for PC vesicles and the vesicle permeabilization increase in the order TUDC < TC < TCDC ≤ TDC.59,60) Increasing PC concentration reduces the membrane permeabilization induced by bile salts.59) Cholesterol incorporation in PC vesicles lowers the binding affinity of taurine-conjugated bile salts and the membrane permeabilization by bile salts.60) Fahey et al. have speculated that bile salt molecules insert horizontally at the surface of phospholipid bilayers, the polar hydroxy groups and hydrophilic side chain of bile salt are exposed to the aqueous medium, and the hydrophobic surface interacts only with the outermost carbons of phospholipid acyl chains.61) On binding to PC vesicles, deoxycholate, ursodeoxycholate and TUDC increase the fluidity of the membrane surface, but not that of the membrane interior, suggesting the location of bile salts near the membrane surface with very limited insertion in the acyl chain region.62) The incorporation of cholesterol into PC vesicles retards the dissolution rate with TC, TUDC or TCDC, and increases the concentration of bile salt required for complete solubilization of vesicles.57,63)
PC/SM vesicles are more sensitive to the micellization with TC than PC vesicles.64,65) The addition of TC to PC/SM vesicles leads to the formation of open vesicles, then to the fusion of bilayer fragments into large open structures coexisting with multilamellar vesicles and thread-like micelles and, finally to the transformation into long thread-like micelles.65) However, the incorporation of cholesterol into PC/SM vesicles profoundly increases the resistance against TC.65) In PC/SM/cholesterol vesicles, deoxycholate or chenodeoxycholate can modulate the fluidity of the liquid disordered phase but not that of the cholesterol and SM-enriched ordered phase.62)
In bile, bile salts, phospholipids and cholesterol form mixed micelles and vesicles, which coexist in dynamic equilibrium with bile salt monomers and simple micelles16,64,66) (Fig. 3). Simple micelles (ca. 3 nm in diameter) can solubilize only small amounts of cholesterol.17) Cholesterol is solubilized in GDC, TDC, GCDC, TCDC, GC, TC, GUDC or TUDC simple micelles at a molar ratio of approximately 1/18, 1/27, 1/35, 1/52, 1/64, 1/90, 1/830 or 1/670, respectively.55) At high bile salt/phospholipid ratios (>4/1), phospholipid molecules are intercalated between bile salt molecules in quasi-spherical mixed micelles (4–8 nm in diameter).15,17) Mixed micelles are thermodynamically stable aggregates. Although mixed micelles are inefficient cholesterol carriers, their capacity to solubilize cholesterol is 1 cholesterol per 3 phospholipid molecules (>12 bile salt molecules), which is greater than that of simple bile salt micelles.15) Vesicles are spherical unilamellar bilayers (40–100 nm in diameter) composed of phospholipids and cholesterol, but poor in bile salts (bile salt/phospholipid ratio of <4/1).15,17) Compared with mixed micelles, bilayer vesicles are much more efficient cholesterol carriers with a solubilizing capacity of approximately 1 cholesterol per phospholipid molecule.15) Bile salt molecules affect the shape of bilayer vesicles by intercalating among phospholipid molecules.15) In human bile, spheroidal micelles, discs, unilamellar vesicles and multilamellar vesicles have been observed by cryo-transmission electron microscopy.67) The bilayer discs (50–100 nm) with bile salts on the perimeter are likely to be intermediates in a micelle-to-vesicle transition.67) Multilamellar vesicles (>500 nm in diameter) may be formed by the fusion of unilamellar vesicles and implicated in the initiation of the cholesterol nucleation.15,17,67) The crystallization of biliary cholesterol is dependent on the bile salt/phospholipid ratio and the hydrophilic–hydrophobic balance of bile salt species in addition to the cholesterol concentration itself.15,17) Wang et al. have constructed an equilibrium bile salt/PC/cholesterol ternary phase diagram and identified five different crystallization pathways.17,68)
Bile salt-induced cell death may be provoked by the increased membrane fluidity and permeability. The cytotoxicities of bile salts have been studied in several cell lines, usually by measuring lactate dehydrogenase release, an index of irreversible injury or necrosis. Heuman et al. have reported that the relative toxicity of different bile salts to primary rat hepatocytes proceed in the following order: TUDC < TC < TDC = TCDC.69) There are no significant differences in the toxicity to rat hepatocytes between glycine- and taurine-conjugated bile salts, and taurine- or glycine-conjugates were less toxic than their unconjugates.69) Martinez-Diez et al. have also shown that the toxicities of chenodeoxycholate and deoxycholate to primary rat hepatocytes were stronger than those of cholate and ursodeoxycholate, and that the conjugation with taurine or glycine markedly reduces the effects of bile salts on cell viability.70) It has been reported that the susceptibilities of Caco-2 cells, a human colon carcinoma cell line, to bile salt-induced damages increase in the order TUDC < TC < TCDC < TDC.71) In the human colon cancer cell line HCT116, deoxycholate and chenodeoxycholate, but not cholate or ursodeoxycholate, induce apoptosis.72) From those findings, the damaging effects of bile salts appear to be related to their hydrophobicities.
We have assessed the cytotoxicities of bile salts by using LLC-PK1 cells, originating from porcine kidney epithelial cells.7) The monolayers of LLC-PK1 cells form apical membranes, which are often used as a model of bile canalicular membranes.29,73) The toxicities to LLC-PK1 cells are in the order TC < GC < cholate < TCDC < GCDC < TDC < GDC < chenodeoxycholate < deoxycholate.7) Furthermore, in distinguishing between unconjugated and conjugated bile salts, there is an inverse correlation between hydrophobicity indices and LD50 values of bile salts for LLC-PK1 cells7) (Fig. 4). We have also compared the bile salt toxicities to the human hepatoma cell line HepG2.8) Similar to LLC-PK1 cells, the order of toxicity to HepG2 cells is cholate < chenodeoxycholate < deoxycholate.8) However, the conjugation of bile salts with taurine or glycine has little or no effect on the toxicity to HepG2 cells.8)
We have determined the LD50 values of unconjugated bile salts (cholate, chenodeoxycholate and deoxycholate) and conjugated bile salts (TC, GC, TCDC, GCDC, TDC and GDC) for LLC-PK1 cells and LLC-PK1 cells stably expressing PEMT (LLC-PK1/PEMT). The hydrophobicity indices, from hydrophilic to hydrophobic, are TC (0.00) < GC (0.07) < cholate (0.13) < TCDC (0.46) < GCDC (0.51) < chenodeoxycholate (0.59) = TDC (0.59) < GDC (0.65) < deoxycholate (0.59). The lines are fitted by single exponential functions. The LD50 values of unconjugated bile salts or conjugated bile salts are inversely correlated with the hydrophobicity indices. Compared with LLC-PK1 cells, LLC-PK1/PEMT cells show lower LD50 values of unconjugated bile salts but higher LD50 values of conjugated bile salts. Reproduced from ref. 7 in slightly modified form with permission from the copyright holder John Wiley and Sons.
The lipid composition of cell membranes is important for the resistance against bile salts. In mammalian cell membranes, PC is the most abundant phospholipid and has a cylindrical shape stabilizing the bilayer membrane structures. PE, the second most abundant phospholipid, has a cone shape forming nonlamellar structures and decreases the activation energy for the negative curvature of the inner leaflet in the microvillus membrane.74,75) PEMT locates in the endoplasmic reticulum (ER) of hepatocytes and catalyzes the generation of PC by the transfer of three methyl groups from S-adenosylmethionine to PE.38) We have proposed that three transmembrane domains of PEMT span the ER membrane, and that the enzyme activity and substrate specificity are modulated by the N-terminal region localized in the ER lumen.76) To clarify the role of PEMT in the cellular resistance to bile salts, we have established LLC-PK1 cells stably expressing PEMT.7) Notably, PEMT expression in LLC-PK1 cells leads to reduced resistance against unconjugated cholate, chenodeoxycholate and deoxycholate, but to increased resistance against taurine- or glycine-conjugated bile salts7) (Fig. 4). The expression of PEMT increases the cellular content of PC, particularly longer acyl chain PC and ether-linked PC, decreases that of PE, but does not change that of SM, which results in reductions of PE/PC and SM/PC ratios. Presumably by decreasing the ratios of PE and SM in the apical membrane, PEMT expression also promotes the resistance to duramycin and lysenin, which bind specifically to PE and SM, respectively.7) Moreover, the expression of PEMT increases the diameter of microvilli on the apical surface of LLC-PK1 cells, which may be caused by the decreased ratio of PE at the inner leaflet of the apical membrane.7) Taken together, these observations suggest that the PEMT-induced resistance to conjugated bile salts occurs due to alterations in the phospholipid composition and/or structures of the apical membranes. Thus, PEMT may have crucial roles in the hepatocyte resistance to bile salt cytotoxicity.
Bile salt monomers and simple micelles, rather than mixed micelles and vesicles, may exert the damaging effects on cell membranes.64) It has been previously suggested that biliary phospholipids and cholesterol represent important cytoprotective factors for hepatocytes against bile salt-induced damage.77) Moschetta et al. have demonstrated that the incorporation of increasing amounts of PC in TC or TDC micelles progressively diminishes the bile salt cytotoxicity to Caco-2 cells.64,78) Velardi et al. have shown that cholesterol alone fails to have any effect on the TC-induced cytotoxicity to Caco-2 cells, but that cholesterol in combination with PC increases the cytotoxicity of TC, which correlates with a cholesterol-induced shift of PC from mixed micelles to vesicles.71)
Recently, we have systematically studied the roles of phospholipids and cholesterol in the protection of hepatocytes from bile salt cytotoxicity.8) The cytotoxicities of cholate, chenodeoxycholate, deoxycholate, TC, GC, TCDC, GCDC, TDC and GDC on HepG2 cells are inhibited with increasing concentration of PC.8) Although cholesterol exerts no direct cytotoxic effect on HepG2 cells, the cytoprotective effects of PC against bile salts are attenuated by the presence of cholesterol.8) Also in the case of primary human hepatocytes, cholesterol suppresses the cytoprotective effects of PC against bile salts.8) The cell association of bile salt molecules is an initial step in the induction of cell death. The amount of cell-associated deoxycholate decreases with increasing PC concentration, but cholesterol facilitates the cell-association of deoxycholate in the presence of PC.8) In the lipid dispersions containing deoxycholate and PC, cholesterol reduces mixed micelles but increases vesicles, deoxycholate simple micelles and deoxycholate monomers.8) Cholesterol is considered to bind strongly to PC, to inhibit the interaction between PC and bile salts, and to stabilize the vesicle structures. Based on those findings, the attenuation of PC cytoprotective effects by cholesterol can be accounted for by the enhanced formation of cytotoxic bile salt simple micelles and monomers (Fig. 5). Thus, biliary PC and cholesterol may differently affect the bile salt toxicity to hepatocytes.
The inclusion of cholesterol within bile salt/phospholipid dispersions suppresses the formation of mixed micelles but promotes that of vesicles, bile salt simple micelles and bile salt monomers. Bile salt simple micelles and monomers, rather than mixed micelles and vesicles, have cytotoxic effects.
In addition to the biliary concentrations of bile salts, those of phospholipids and cholesterol are important for the cytotoxicity of bile salts. The biliary concentrations of bile salts, phospholipids and cholesterol are largely dependent on the efflux activities of ABCB11, ABCB4 and ABCG5/ABCG8, respectively. However, the regulatory mechanisms of the biliary secretions of bile salts, phospholipids and cholesterol remain poorly understood. Ursodeoxycholic acid is widely used for the treatment of cholestatic liver diseases, despite limited benefits.9,10) Elucidation of the molecular mechanisms underlying the bile salt, phospholipid and cholesterol secretions mediated by ABC transporters will be useful in developing new therapies for cholestatic liver diseases.
We are grateful to John Wiley and Sons for permission to reproduce Fig. 4 in slightly modified form. This work was supported in part by the PRIME from Japan Agency for Medical Research and Development (AMED) and by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers JP15H05660 and JP18K14918.
The authors declare no conflict of interest.