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Biliary cholesterol secretion: more lessons from plants?
Journal of Hepatology 38 (2003) 843–846 www.elsevier.com/locate/jhep
Editorial
Biliary cholesterol secretion: more lessons from plants? Bruno Stieger* Division of Clinical Pharmacology and Toxicology, Department of Medicine, University Hospital, 8091 Zurich, Switzerland
See Article, pages 710 –716
Plasma cholesterol levels positively correlate with atherosclerosis, which is a major health care problem in Western societies. Plasma cholesterol level is determined by the balance of dietary cholesterol intake via the intestine, its distribution to the organs, cholesterol biosynthesis, use of cholesterol for biosynthesis of steroids and bile salts and biliary cholesterol secretion [1]. The liver is the only organ that can excrete significant amounts of cholesterol, either by converting it biosynthetically into bile salts or by secreting it as unesterified cholesterol into bile [1,2]. From there, cholesterol enters the small intestine, where it is partially reabsorbed and partially excreted via feces. Cholesterol absorption in the intestine and cholesterol secretion into bile requires bile salts. Hence, hepatic secretion of cholesterol and enterohepatic circulation of bile acids are key determinants of plasma cholesterol level. Bile acids undergo efficient enterohepatic circulation with a fecal loss of only about 0.5 g per day [3]. This loss is compensated for by hepatic de novo synthesis starting from cholesterol [1,2]. Presumably, most of the transport systems involved in enterohepatic circulation of bile acids have been identified and characterized at the molecular level [3]. In contrast, the molecular mechanisms of cholesterol absorption in the intestine and of cholesterol secretion into bile are less well understood. The basic principle of biliary cholesterol solubility in bile [4] and of biliary lipid secretion have been known for a long time [5]: biliary phospholipids (predominantly phosphatidylcholine), secreted by the multidrug resistance protein 2 MDR2 (ABCB4) and bile salts secreted by the bile salt export pump BSEP (ABCB11) provide together the vehicle for cholesterol solubilization. The biliary secretion of lipids depends on bile salt secretion and, in addition, efficient biliary cholesterol secretion requires ongoing biliary phospholipid secretion [6]. * Tel.: þ41-1-255-2068; fax: þ 41-1-255-4411. E-mail address: [email protected] (B. Stieger).
Currently, rapidly emerging new findings on biliary cholesterol secretion are greatly enhancing our understanding of the molecular mechanisms of cholesterol output into bile. Important insight into these mechanisms was gained from the identification of the genes responsible for sitosterolemia. This rare, autosomal recessively inherited disease manifests itself by the presence of tendon xanthomas, premature coronary artery and atheroslerotic disease, hemolytic episodes, arthralgias and arthritis and is characterized by highly elevated levels of plant sterols in plasma [7]. Under normal circumstances, plant sterols, which are about equal to the amount of cholesterol in our diet, are only minimally absorbed in the small intestine and efficiently removed from portal blood plasma and excreted into bile by hepatocytes. In addition to plant sterols, patients with sitosterolemia may have moderately elevated plasma cholesterol levels [7]. Genetic studies in humans affected with sitosterolemia identified the two genes ABCG5 and ABCG8 as important determinants of biliary sitosterol and cholesterol excretion. They form an adjacent pair of genes on chromosome 2p21 [8,9]. Patients with sitosterolemia have mutations in the ABCG5 or the ABCG8 gene. These two genes encode for the ATP-binding cassette (ABC) transporters ABCG5 and ABCG8, which are ABC half transporters with each having six predicted transmembrane spanning domains. Expression studies of epitope-tagged ABCG5 and ABCG8 in different non-polarized and polarized cell lines revealed that ABCG5 and ABCG8 need to form a heterodimer in the endoplasmic reticulum to reach the cell surface [10]. In the polarized WIF-B hepatocyte cell line, only heterodimers of ABCG5 and ABCG8 localize to the canalicular plasma membrane domain [10]. This finding is compatible with the observation that mutations in either ABCG5 or ABCG8 lead to the clinical phenotype of sitosterolemia. Overexpression of human ABCG5 and ABCG8 in transgenic mice lead to a reduced fractional absorption of dietary cholesterol in the
0168-8278/03/$30.00 q 2003 Published by Elsevier Science B.V. on behalf of the European Association for the Study of the Liver. doi:10.1016/S0168-8278(03)00194-6
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B. Stieger / Journal of Hepatology 38 (2003) 843–846
intestine and to a more than 5-fold increase in hepatic cholesterol secretion into bile [11]. Plasma levels of cholesterol were slightly reduced, while plant sterol levels were markedly reduced. These changes were paralleled by a strongly increased fecal excretion of neutral sterols [11]. Together, these data provide strong evidence for ABCG5 and ABCG8 being important for both, mediating intestinal (back) efflux of sitosterol and other plant sterols and hepatocellular secretion of cholesterol. Using a reverse approach by disrupting Abcg5 and Abcg8 in mice, biliary cholesterol secretion was reduced by about 90% concomitant with a decreased hepatic cholesterol and increased hepatic sitosterol level in animals on low cholesterol diet [12]. Plasma sitosterol level was elevated about 30-fold in the knockout mice on low cholesterol diet along with an increased fractional absorption of plant sterols in the intestine. In contrast, fractional intestinal cholesterol absorption was not affected by the inactivation of Abcg5 and Abcg8. These findings further support that ABCG5/ Abcg5 and ABCG8/Abcg8 are directly involved in canalicular secretion of cholesterol and sitosterol and that they prevent plant sterols from entering the systemic circulation from the lumen of the intestine. Therefore, there is now firm evidence for the complete molecular separation of the biliary secretory pathways for phospholipids and cholesterol [13]. The four ABC transporters ABCG5 and ABCG8, MDR2 and BSEP are key components in canalicular lipid secretion and mutations in their genes lead to various forms of disease [4]. However, it is interesting to note that feeding mice with a diet high in cholesterol increased biliary cholesterol secretion in these knockout animals, suggesting that additional cholesterol secretory mechanisms might still exist at the hepatocanalicular membrane [12]. In this issue of the Journal, Kosters et al. investigated the relation between hepatic Abcg5 and Abcg8 expression levels in mice and biliary cholesterol secretion [14]. They did so by using mouse models with various biliary cholesterol secretion rates. The key finding of their study is the observation of a correlation between biliary cholesterol output and Abcg5/Abcg8 expression. If cholesterol output was normalized for phospholipid output, the relation with Abcg5/Abcg8 expression became linear. Most interestingly however, mice fed diosgenin, a plant-derived sapogenin, had a massive stimulation of cholesterol secretion without changing Abcg5/Abcg8 expression levels. Additionally, the study shows that hepatic and intestinal Abcg5/Abcg8 expression is not regulated in parallel. Hence, although the identification of ABCG5/Abcg5 and ABCG8/ Abcg8 has considerably advanced the understanding of canalicular cholesterol secretion into bile, the study of Kosters et al. underlines that there is need for more research in this area. In particular, the data from Kosters et al. on biliary cholesterol hypersecretion induced by diosgenin feeding without concomitant upregulation of Abcg5/Abcg8 [14]
could be interpreted as evidence for the presence of a yet unidentified additional system or mechanism for cholesterol secretion in the canalicular plasma membrane or along the biliary tract. It is interesting to note that an earlier study using gel-permeation chromatography found in bile with moderate cholesterol level from normal rats a single peak of cholesterol eluting [15]. In bile with high cholesterol concentrations (. 0.9 mM) from normal rats a second, small peak occurring with the void volume was eluted, indicating the presence of larger biliary complexes containing cholesterol [15]. When bile from disogenin-fed rats was analyzed, this early peak corresponding to the void volume was prominent and its height positively correlated with increasing biliary cholesterol concentrations. In diosgeninfed rats, the secretion of the canalicular enzyme alkaline phosphodiesterase I was significantly lower than in controls, indicating that a general damage was not imposed on the canalicular plasma membrane by diosgenin feeding [15]. Furthermore, Thewles et al. also demonstrated that increased cholesterol secretion in the diosgenin treated animals was strictly dependent on bile salt secretion. A later study morphologically characterized the livers of diosgenin treated rats and found a massive loss of canalicular microvilli in comparison to control livers [16]. The appearance of canaliculi of diosgenin treated rats resembles a cholestatic phenotype, even though bile flow, bile salt and phospholipid secretion were normal [16]. Furthermore Accatino et al. reported that additional treatment with ethinylestradiol of diosgenin-fed rats completely abolished the stimulation of cholesterol secretion. Indeed, bile flow and cholesterol secretion were impaired to levels lower than in untreated controls and were as low as in estradiol treated rats [16]. Using electronmicroscopic analysis of bile of diosgenin treated animals, multiple curved parallel sheets in both the form of vesicles and curved stacked lamellae were observed [16]. Such structures were absent in control bile. It seems likely that these large biliary structures observed in bile of diosgenin treated rats [16] correspond to the fraction eluting in the void volume of gel permeation chromatography [15]. Since the occurrence of the early peak needs a threshold concentration of cholesterol [15], the formation of such complexes could occur after secretion of cholesterol into bile. Diosgenin increases hepatic cholesterol synthesis in rats [17] and thus could lead to an increased hepatocellular supply of cholesterol to the canalicular membrane. This could potentially affect the cholesterol content of the canalicular plasma membrane. Analysis of canalicular membranes of diosgenin treated rats revealed both, unchanged [18,19] and increased cholesterol contents [16, 20]. These conflicting results may be explained by methodological differences. In addition, the source of biliary cholesterol is predominantly derived from high density lipoprotein associated cholesterol and not from hepatocellular de novo synthesis [2,21]. Taken together, these data do neither support nor exclude the presence of an additional canalicular cholesterol transport system.
B. Stieger / Journal of Hepatology 38 (2003) 843–846
Besides its effect on the liver, diosgenin is secreted into bile [22] and it also decreases intestinal cholesterol absorption in rats [17]. So far, the exact subcellular localization of ABCG5/Abcg5 and ABCG8/ Abcg8 in liver and in intestine has not yet been determined in vivo. However, it is tempting to speculate that inhibition of intestinal cholesterol absorption by diosgenin may be due to an activation of Abcg5/Abcg8 by a yet unknown mechanism with subsequent reduction of intestinal cholesterol absorption. A similar mechanism could lead to the activation of Abcg5/Abcg8 in the canalicular membrane concomitant with increased biliary cholesterol secretion. Last but not least, if Abcg5/Abcg8 were also present in cholangiocytes, the same mechanism could prevent reabsorption of cholesterol by cholangiocytes and hence lead to a further apparent stimulation of biliary cholesterol secretion. This latter view is supported by an observation in dogs, where infusion of tauroursodeoxycholate stimulated biliary cholesterol secretion more than biliary phospholipid secretion [23]. As an explanation for this observation, tauroursodeoxycholate-mediated lowering of cholesterol reabsorption along the biliary tree was discussed. Two recent publications provide evidence for genes other than Abcg5/Abcg8 to be implicated in cholesterol transport. A screen of 20 different mouse strains identified a large number of polymorphic variants of Abcg5/Abcg8 [24]. Strains reported to show major differences in intestinal cholesterol absorption showed no correlation with genomic variations in Abcg5/Abcg8 [24]. In another study, intercrossing of mice with different campesterol and sitosterol plasma levels linked two loci on chromosomes 14 and 2 distinct from Abcg5/Abcg8 to the regulation of plasma levels of plant sterols [25]. Even though these findings may not lead to the identification of genes for additional cholesterol and/or plant sterol transporters, one should remain open for novel putative cholesterol/plant sterol transporters. It is evident that biliary cholesterol secretion is dependent on the correct functional interplay of several canalicular ABC transporters in hepatocytes. First evidence is now available, which highlights the importance of protein-protein interaction for canalicular bile formation: disruption of the PDZK1 gene in mice results in elevated plasma cholesterol levels [26]. Similarly, inactivation of the radixin gene in mice causes conjugated hyperbilirubinemia with loss of Mrp2 from the canalicular membrane [27]. These results point to novel aspects in canalicular bile formation and its regulation. Additionally, one major challenge in understanding canalicular cholesterol secretion remains: evidence needs to be provided by heterologous expression of ABCG5/Abcg5 and ABCG8/Abcg8 that these proteins are indeed cholesterol transporting ABC transporters.
845
References [1] Lu K, Lee MH, Patel SB. Dietary cholesterol absorption; more than just bile. Trends Endocrinol Metab 2001;12:314– 20. [2] Cohen DE. Hepatocellular transport and secretion of biliary lipids. Curr Opin Lipidol 1999;10:295–302. [3] Meier PJ, Stieger B. Bile salt transporters. Annu Rev Physiol 2002;64: 635–61. [4] Small DM. Role of ABC transporters in secretion of cholesterol from liver into bile. Proc Natl Acad Sci USA 2003;100:4 –6. [5] Coleman R. Biochemistry of bile secretion. Biochem J 1987;244: 249–61. [6] Oude Elferink RPM, Groen AK. Mechanisms of biliary lipid secretion and their role in lipid homeostasis. Semin Liver Dis 2000;20: 293–305. [7] Lee MH, Lu K, Patel SB. Genetic basis of sitosterolemia. Curr Opin Lipidol 2001;12:141 –9. [8] Berge KE, Tian H, Graf GA, Yu L, Grishin NV, Schultz J, et al. Accumulation of dietary cholesterol in sitosterolemia caused by mutations in adjacent ABC transporters. Science 2000;290:1771–5. [9] Lee MH, Lu K, Hazard S, Yu H, Shulenin S, Hidaka H, et al. Identification of a gene, ABCG5, important in the regulation of dietary cholesterol absorption. Nat Genet 2001;27:79–83. [10] Graf GA, Li WP, Gerard RD, Gelissen I, White A, Cohen JC, et al. Coexpression of ATP-binding cassette proteins ABCG5 and ABCG8 permits their transport to the apical surface. J Clin Invest 2002;110: 659–69. [11] Yu L, Li Hawkins J, Hammer RE, Berge KE, Horton JD, Cohen JC, et al. Overexpression of ABCG5 and ABCG8 promotes biliary cholesterol secretion and reduces fractional absorption of dietary cholesterol. J Clin Invest 2002;110:671 –80. [12] Yu L, Hammer RE, Li Hawkins J, Von Bergmann K, Lutjohann D, Cohen JC, et al. Disruption of Abcg5 and Abcg8 in mice reveals their crucial role in biliary cholesterol secretion. Proc Natl Acad Sci USA 2002;99:16237–42. [13] Wagner CI, Trotman BW, Soloway RD. Kinetic analysis of biliary lipid excretion in man and dog. J Clin Invest 1976;57:473 –7. [14] Kosters A, Frijters RJJM, Schaap FG, Vink E, Plo¨sch T, Ottenhoff R, et al. Relation between hepatic expression of ATP-binding cassette transporters G5 and G8 and biliary cholesterol secretion in mice. J Hepatol 2003;38:710– 6. [15] Thewles A, Parslow RA, Coleman R. Effect of diosgenin on biliary cholesterol transport in the rat. Biochem J 1993;291:793–8. [16] Accatino L, Pizarro M, Solis N, Koenig CS. Effects of diosgenin, a plant-derived steroid, on bile secretion and hepatocellular cholestasis induced by estrogens in the rat. Hepatology 1998;28:129–40. [17] Cayen MN, Dvornik D. Effect of diosgenin on lipid metabolism in rats. J Lipid Res 1979;20:162–74. [18] Nibbering CP, Groen AK, Ottenhoff R, Brouwers JF, vanBerge Henegouwen GP, van Erpecum KJ. Regulation of biliary cholesterol secretion is independent of hepatocyte canalicular membrane lipid composition: a study in the diosgenin-fed rat model. J Hepatol 2001; 35:164–9. [19] Roman ID, Thewles A, Coleman R. Fractionation of livers following diosgenin treatment to elevate biliary cholesterol. Biochim Biophys Acta 1995;1255:77 –81. [20] Amigo L, Mendoza H, Zanlungo S, Miquel JF, Rigotti A, Gonzalez S, et al. Enrichment of canalicular membrane with cholesterol and sphingomyelin prevents bile salt-induced hepatic damage. J Lipid Res 1999;40:533 –42. [21] Carey MC. Homing-in on the origin of biliary steroids. Gut 1997;41: 721–2. [22] Cayen MN, Ferdinandi ES, Greselin E, Dvornik D. Studies on the disposition of diosgenin in rats, dogs, monkeys and man. Atherosclerosis 1979;33:71–87. [23] Gilmore IT, Barnhart JL, Hofmann AF, Erlinger S. Effects of
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B. Stieger / Journal of Hepatology 38 (2003) 843–846
individual taurine-conjugated bile acids on biliary lipid secretion and sucrose clearance in the unanesthetized dog. Am J Physiol 1982;242: G40–6. [24] Lu K, Lee MH, Yu H, Zhou Y, Sandell SA, Salen G, et al. Molecular cloning, genomic organization, genetic variations, and characterization of murine sterolin genes Abcg5 and Abcg8. J Lipid Res 2002;43: 565–78. [25] Sehayek E, Duncan EM, Lutjohann D, Von Bergmann K, Ono JG, Batta AK, et al. Loci on chromosomes 14 and 2, distinct from
ABCG5/ABCG8, regulate plasma plant sterol levels in a C57BL/ 6J £ CASA/Rk intercross. Proc Natl Acad Sci USA 2002;99: 16215–9. [26] Kocher O, Pal R, Roberts M, Cirovic C, Gilchrist A. Targeted disruption of the PDZK1 gene by homologous recombination. Mol Cell Biol 2003;23:1175–80. [27] Kikuchi S, Hata M, Fukumoto K, Yamane Y, Matsui T, Tamura A, et al. Radixin deficiency causes conjugated hyperbilirubinemia with loss of Mrp2 from bile canalicular membranes. Nat Genet 2002;31:320–5.
Editorial
Biliary cholesterol secretion: more lessons from plants? Bruno Stieger* Division of Clinical Pharmacology and Toxicology, Department of Medicine, University Hospital, 8091 Zurich, Switzerland
See Article, pages 710 –716
Plasma cholesterol levels positively correlate with atherosclerosis, which is a major health care problem in Western societies. Plasma cholesterol level is determined by the balance of dietary cholesterol intake via the intestine, its distribution to the organs, cholesterol biosynthesis, use of cholesterol for biosynthesis of steroids and bile salts and biliary cholesterol secretion [1]. The liver is the only organ that can excrete significant amounts of cholesterol, either by converting it biosynthetically into bile salts or by secreting it as unesterified cholesterol into bile [1,2]. From there, cholesterol enters the small intestine, where it is partially reabsorbed and partially excreted via feces. Cholesterol absorption in the intestine and cholesterol secretion into bile requires bile salts. Hence, hepatic secretion of cholesterol and enterohepatic circulation of bile acids are key determinants of plasma cholesterol level. Bile acids undergo efficient enterohepatic circulation with a fecal loss of only about 0.5 g per day [3]. This loss is compensated for by hepatic de novo synthesis starting from cholesterol [1,2]. Presumably, most of the transport systems involved in enterohepatic circulation of bile acids have been identified and characterized at the molecular level [3]. In contrast, the molecular mechanisms of cholesterol absorption in the intestine and of cholesterol secretion into bile are less well understood. The basic principle of biliary cholesterol solubility in bile [4] and of biliary lipid secretion have been known for a long time [5]: biliary phospholipids (predominantly phosphatidylcholine), secreted by the multidrug resistance protein 2 MDR2 (ABCB4) and bile salts secreted by the bile salt export pump BSEP (ABCB11) provide together the vehicle for cholesterol solubilization. The biliary secretion of lipids depends on bile salt secretion and, in addition, efficient biliary cholesterol secretion requires ongoing biliary phospholipid secretion [6]. * Tel.: þ41-1-255-2068; fax: þ 41-1-255-4411. E-mail address: [email protected] (B. Stieger).
Currently, rapidly emerging new findings on biliary cholesterol secretion are greatly enhancing our understanding of the molecular mechanisms of cholesterol output into bile. Important insight into these mechanisms was gained from the identification of the genes responsible for sitosterolemia. This rare, autosomal recessively inherited disease manifests itself by the presence of tendon xanthomas, premature coronary artery and atheroslerotic disease, hemolytic episodes, arthralgias and arthritis and is characterized by highly elevated levels of plant sterols in plasma [7]. Under normal circumstances, plant sterols, which are about equal to the amount of cholesterol in our diet, are only minimally absorbed in the small intestine and efficiently removed from portal blood plasma and excreted into bile by hepatocytes. In addition to plant sterols, patients with sitosterolemia may have moderately elevated plasma cholesterol levels [7]. Genetic studies in humans affected with sitosterolemia identified the two genes ABCG5 and ABCG8 as important determinants of biliary sitosterol and cholesterol excretion. They form an adjacent pair of genes on chromosome 2p21 [8,9]. Patients with sitosterolemia have mutations in the ABCG5 or the ABCG8 gene. These two genes encode for the ATP-binding cassette (ABC) transporters ABCG5 and ABCG8, which are ABC half transporters with each having six predicted transmembrane spanning domains. Expression studies of epitope-tagged ABCG5 and ABCG8 in different non-polarized and polarized cell lines revealed that ABCG5 and ABCG8 need to form a heterodimer in the endoplasmic reticulum to reach the cell surface [10]. In the polarized WIF-B hepatocyte cell line, only heterodimers of ABCG5 and ABCG8 localize to the canalicular plasma membrane domain [10]. This finding is compatible with the observation that mutations in either ABCG5 or ABCG8 lead to the clinical phenotype of sitosterolemia. Overexpression of human ABCG5 and ABCG8 in transgenic mice lead to a reduced fractional absorption of dietary cholesterol in the
0168-8278/03/$30.00 q 2003 Published by Elsevier Science B.V. on behalf of the European Association for the Study of the Liver. doi:10.1016/S0168-8278(03)00194-6
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B. Stieger / Journal of Hepatology 38 (2003) 843–846
intestine and to a more than 5-fold increase in hepatic cholesterol secretion into bile [11]. Plasma levels of cholesterol were slightly reduced, while plant sterol levels were markedly reduced. These changes were paralleled by a strongly increased fecal excretion of neutral sterols [11]. Together, these data provide strong evidence for ABCG5 and ABCG8 being important for both, mediating intestinal (back) efflux of sitosterol and other plant sterols and hepatocellular secretion of cholesterol. Using a reverse approach by disrupting Abcg5 and Abcg8 in mice, biliary cholesterol secretion was reduced by about 90% concomitant with a decreased hepatic cholesterol and increased hepatic sitosterol level in animals on low cholesterol diet [12]. Plasma sitosterol level was elevated about 30-fold in the knockout mice on low cholesterol diet along with an increased fractional absorption of plant sterols in the intestine. In contrast, fractional intestinal cholesterol absorption was not affected by the inactivation of Abcg5 and Abcg8. These findings further support that ABCG5/ Abcg5 and ABCG8/Abcg8 are directly involved in canalicular secretion of cholesterol and sitosterol and that they prevent plant sterols from entering the systemic circulation from the lumen of the intestine. Therefore, there is now firm evidence for the complete molecular separation of the biliary secretory pathways for phospholipids and cholesterol [13]. The four ABC transporters ABCG5 and ABCG8, MDR2 and BSEP are key components in canalicular lipid secretion and mutations in their genes lead to various forms of disease [4]. However, it is interesting to note that feeding mice with a diet high in cholesterol increased biliary cholesterol secretion in these knockout animals, suggesting that additional cholesterol secretory mechanisms might still exist at the hepatocanalicular membrane [12]. In this issue of the Journal, Kosters et al. investigated the relation between hepatic Abcg5 and Abcg8 expression levels in mice and biliary cholesterol secretion [14]. They did so by using mouse models with various biliary cholesterol secretion rates. The key finding of their study is the observation of a correlation between biliary cholesterol output and Abcg5/Abcg8 expression. If cholesterol output was normalized for phospholipid output, the relation with Abcg5/Abcg8 expression became linear. Most interestingly however, mice fed diosgenin, a plant-derived sapogenin, had a massive stimulation of cholesterol secretion without changing Abcg5/Abcg8 expression levels. Additionally, the study shows that hepatic and intestinal Abcg5/Abcg8 expression is not regulated in parallel. Hence, although the identification of ABCG5/Abcg5 and ABCG8/ Abcg8 has considerably advanced the understanding of canalicular cholesterol secretion into bile, the study of Kosters et al. underlines that there is need for more research in this area. In particular, the data from Kosters et al. on biliary cholesterol hypersecretion induced by diosgenin feeding without concomitant upregulation of Abcg5/Abcg8 [14]
could be interpreted as evidence for the presence of a yet unidentified additional system or mechanism for cholesterol secretion in the canalicular plasma membrane or along the biliary tract. It is interesting to note that an earlier study using gel-permeation chromatography found in bile with moderate cholesterol level from normal rats a single peak of cholesterol eluting [15]. In bile with high cholesterol concentrations (. 0.9 mM) from normal rats a second, small peak occurring with the void volume was eluted, indicating the presence of larger biliary complexes containing cholesterol [15]. When bile from disogenin-fed rats was analyzed, this early peak corresponding to the void volume was prominent and its height positively correlated with increasing biliary cholesterol concentrations. In diosgeninfed rats, the secretion of the canalicular enzyme alkaline phosphodiesterase I was significantly lower than in controls, indicating that a general damage was not imposed on the canalicular plasma membrane by diosgenin feeding [15]. Furthermore, Thewles et al. also demonstrated that increased cholesterol secretion in the diosgenin treated animals was strictly dependent on bile salt secretion. A later study morphologically characterized the livers of diosgenin treated rats and found a massive loss of canalicular microvilli in comparison to control livers [16]. The appearance of canaliculi of diosgenin treated rats resembles a cholestatic phenotype, even though bile flow, bile salt and phospholipid secretion were normal [16]. Furthermore Accatino et al. reported that additional treatment with ethinylestradiol of diosgenin-fed rats completely abolished the stimulation of cholesterol secretion. Indeed, bile flow and cholesterol secretion were impaired to levels lower than in untreated controls and were as low as in estradiol treated rats [16]. Using electronmicroscopic analysis of bile of diosgenin treated animals, multiple curved parallel sheets in both the form of vesicles and curved stacked lamellae were observed [16]. Such structures were absent in control bile. It seems likely that these large biliary structures observed in bile of diosgenin treated rats [16] correspond to the fraction eluting in the void volume of gel permeation chromatography [15]. Since the occurrence of the early peak needs a threshold concentration of cholesterol [15], the formation of such complexes could occur after secretion of cholesterol into bile. Diosgenin increases hepatic cholesterol synthesis in rats [17] and thus could lead to an increased hepatocellular supply of cholesterol to the canalicular membrane. This could potentially affect the cholesterol content of the canalicular plasma membrane. Analysis of canalicular membranes of diosgenin treated rats revealed both, unchanged [18,19] and increased cholesterol contents [16, 20]. These conflicting results may be explained by methodological differences. In addition, the source of biliary cholesterol is predominantly derived from high density lipoprotein associated cholesterol and not from hepatocellular de novo synthesis [2,21]. Taken together, these data do neither support nor exclude the presence of an additional canalicular cholesterol transport system.
B. Stieger / Journal of Hepatology 38 (2003) 843–846
Besides its effect on the liver, diosgenin is secreted into bile [22] and it also decreases intestinal cholesterol absorption in rats [17]. So far, the exact subcellular localization of ABCG5/Abcg5 and ABCG8/ Abcg8 in liver and in intestine has not yet been determined in vivo. However, it is tempting to speculate that inhibition of intestinal cholesterol absorption by diosgenin may be due to an activation of Abcg5/Abcg8 by a yet unknown mechanism with subsequent reduction of intestinal cholesterol absorption. A similar mechanism could lead to the activation of Abcg5/Abcg8 in the canalicular membrane concomitant with increased biliary cholesterol secretion. Last but not least, if Abcg5/Abcg8 were also present in cholangiocytes, the same mechanism could prevent reabsorption of cholesterol by cholangiocytes and hence lead to a further apparent stimulation of biliary cholesterol secretion. This latter view is supported by an observation in dogs, where infusion of tauroursodeoxycholate stimulated biliary cholesterol secretion more than biliary phospholipid secretion [23]. As an explanation for this observation, tauroursodeoxycholate-mediated lowering of cholesterol reabsorption along the biliary tree was discussed. Two recent publications provide evidence for genes other than Abcg5/Abcg8 to be implicated in cholesterol transport. A screen of 20 different mouse strains identified a large number of polymorphic variants of Abcg5/Abcg8 [24]. Strains reported to show major differences in intestinal cholesterol absorption showed no correlation with genomic variations in Abcg5/Abcg8 [24]. In another study, intercrossing of mice with different campesterol and sitosterol plasma levels linked two loci on chromosomes 14 and 2 distinct from Abcg5/Abcg8 to the regulation of plasma levels of plant sterols [25]. Even though these findings may not lead to the identification of genes for additional cholesterol and/or plant sterol transporters, one should remain open for novel putative cholesterol/plant sterol transporters. It is evident that biliary cholesterol secretion is dependent on the correct functional interplay of several canalicular ABC transporters in hepatocytes. First evidence is now available, which highlights the importance of protein-protein interaction for canalicular bile formation: disruption of the PDZK1 gene in mice results in elevated plasma cholesterol levels [26]. Similarly, inactivation of the radixin gene in mice causes conjugated hyperbilirubinemia with loss of Mrp2 from the canalicular membrane [27]. These results point to novel aspects in canalicular bile formation and its regulation. Additionally, one major challenge in understanding canalicular cholesterol secretion remains: evidence needs to be provided by heterologous expression of ABCG5/Abcg5 and ABCG8/Abcg8 that these proteins are indeed cholesterol transporting ABC transporters.
845
References [1] Lu K, Lee MH, Patel SB. Dietary cholesterol absorption; more than just bile. Trends Endocrinol Metab 2001;12:314– 20. [2] Cohen DE. Hepatocellular transport and secretion of biliary lipids. Curr Opin Lipidol 1999;10:295–302. [3] Meier PJ, Stieger B. Bile salt transporters. Annu Rev Physiol 2002;64: 635–61. [4] Small DM. Role of ABC transporters in secretion of cholesterol from liver into bile. Proc Natl Acad Sci USA 2003;100:4 –6. [5] Coleman R. Biochemistry of bile secretion. Biochem J 1987;244: 249–61. [6] Oude Elferink RPM, Groen AK. Mechanisms of biliary lipid secretion and their role in lipid homeostasis. Semin Liver Dis 2000;20: 293–305. [7] Lee MH, Lu K, Patel SB. Genetic basis of sitosterolemia. Curr Opin Lipidol 2001;12:141 –9. [8] Berge KE, Tian H, Graf GA, Yu L, Grishin NV, Schultz J, et al. Accumulation of dietary cholesterol in sitosterolemia caused by mutations in adjacent ABC transporters. Science 2000;290:1771–5. [9] Lee MH, Lu K, Hazard S, Yu H, Shulenin S, Hidaka H, et al. Identification of a gene, ABCG5, important in the regulation of dietary cholesterol absorption. Nat Genet 2001;27:79–83. [10] Graf GA, Li WP, Gerard RD, Gelissen I, White A, Cohen JC, et al. Coexpression of ATP-binding cassette proteins ABCG5 and ABCG8 permits their transport to the apical surface. J Clin Invest 2002;110: 659–69. [11] Yu L, Li Hawkins J, Hammer RE, Berge KE, Horton JD, Cohen JC, et al. Overexpression of ABCG5 and ABCG8 promotes biliary cholesterol secretion and reduces fractional absorption of dietary cholesterol. J Clin Invest 2002;110:671 –80. [12] Yu L, Hammer RE, Li Hawkins J, Von Bergmann K, Lutjohann D, Cohen JC, et al. Disruption of Abcg5 and Abcg8 in mice reveals their crucial role in biliary cholesterol secretion. Proc Natl Acad Sci USA 2002;99:16237–42. [13] Wagner CI, Trotman BW, Soloway RD. Kinetic analysis of biliary lipid excretion in man and dog. J Clin Invest 1976;57:473 –7. [14] Kosters A, Frijters RJJM, Schaap FG, Vink E, Plo¨sch T, Ottenhoff R, et al. Relation between hepatic expression of ATP-binding cassette transporters G5 and G8 and biliary cholesterol secretion in mice. J Hepatol 2003;38:710– 6. [15] Thewles A, Parslow RA, Coleman R. Effect of diosgenin on biliary cholesterol transport in the rat. Biochem J 1993;291:793–8. [16] Accatino L, Pizarro M, Solis N, Koenig CS. Effects of diosgenin, a plant-derived steroid, on bile secretion and hepatocellular cholestasis induced by estrogens in the rat. Hepatology 1998;28:129–40. [17] Cayen MN, Dvornik D. Effect of diosgenin on lipid metabolism in rats. J Lipid Res 1979;20:162–74. [18] Nibbering CP, Groen AK, Ottenhoff R, Brouwers JF, vanBerge Henegouwen GP, van Erpecum KJ. Regulation of biliary cholesterol secretion is independent of hepatocyte canalicular membrane lipid composition: a study in the diosgenin-fed rat model. J Hepatol 2001; 35:164–9. [19] Roman ID, Thewles A, Coleman R. Fractionation of livers following diosgenin treatment to elevate biliary cholesterol. Biochim Biophys Acta 1995;1255:77 –81. [20] Amigo L, Mendoza H, Zanlungo S, Miquel JF, Rigotti A, Gonzalez S, et al. Enrichment of canalicular membrane with cholesterol and sphingomyelin prevents bile salt-induced hepatic damage. J Lipid Res 1999;40:533 –42. [21] Carey MC. Homing-in on the origin of biliary steroids. Gut 1997;41: 721–2. [22] Cayen MN, Ferdinandi ES, Greselin E, Dvornik D. Studies on the disposition of diosgenin in rats, dogs, monkeys and man. Atherosclerosis 1979;33:71–87. [23] Gilmore IT, Barnhart JL, Hofmann AF, Erlinger S. Effects of
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