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The coming of age of our understanding of the enterohepatic circulation of bile salts
The American Journal of Surgery 185 (2003) 168 –172
Review
The coming of age of our understanding of the enterohepatic circulation of bile salts Richard N. Redinger, M.D.* Department of Medicine, University of Louisville, 530 South Jackson St., 3rd Floor, Louisville, KY 40292, USA Manuscript received April 22, 2002; revised manuscript August 12, 2002
Abstract Recent advances in molecular biology have greatly accelerated knowledge relating to the significance of the enterohepatic circulation of bile salts. This review highlights the role that both oxysterols and bile salts play as ligands which, when bound to nuclear hormone receptors, activate transcription factors that set into play feed-forward catabolism of cholesterol to bile salts and feedback control of bile acid synthesis. The nuclear hormone receptors, liver X receptor (LXR) and farnesoid X receptor (FXR) both combined as heterodimers with retinoid X receptor and with oxysterols and bile salts, respectively as their ligands, initiate powerful genetic controls over cholesterol and bile acid homeostatic mechanisms. LXR/RXR signals molecular control of feed-forward catabolism of cholesterol to bile acids while FXR/RXR initiates feedback control of bile acid synthesis. An additional nuclear hormone receptor, small heterodimer partner (SHP), is required to inhibit the competence factor, liver receptor homolog-1 to achieve repression of bile acid synthesis in the liver and in so doing SHP autoregulates its own function. Additionally, while bile acid synthesis is repressed, pool size is preserved by the action of FXR/RXR at both hepatic and intestinal levels, which genetically signals enhanced hepatocyte bile salt transport by the bile salt export pump (BSEP) and the ileal bile acid binding protein (IBABP) for ileal reabsorption. During activation of cholesterol catabolism, LXR/RXR enhances reverse cholesterol transport by increasing cholesterol efflux via the ABC-1 transporter from extrahepatic cells. This cholesterol is then taken up by high-density lipoprotein (HDL) and transported back to the liver for further cholesterol catabolism and elimination in bile. The genetic coordination of nuclear hormone receptor function within the territory of the enterohepatic of bile salts allows for normal cholesterol and bile acid homeostasis thereby preventing atherosclerosis. © 2003 Excerpta Medica Inc. All rights reserved. Keywords: Nuclear hormone receptors; Bile salt; Cholesterol homeostasis
Recent advances in molecular biology have greatly accelerated knowledge relating to the significance of the enterohepatic circulation of bile salts [1]. (Bile acids are synthesized with a terminal acidic group, but when conjugated with glycine or taurine and at physiological pH, they are negatively charged and exist in their anion form. In this review, the term “bile acid” denotes synthesis whereas as “bile salts” denotes their physiological state at normal cellular pH.) The great hope that bile salts would provide an effective medical treatment for cholesterol gallstone disease had not fulfilled enthusiastic expectations [2] and with the widespread practice of safe laparoscopic cholecystectomy, bile acid research had languished for almost two decades awaiting further advances in molecular biology. The clinical significance of preserving the bile salt pool for lipid absorp-
* Corresponding author. Tel.: ⫹1-502-852-5241; fax: ⫹1-502-8526233.
tion [3] and its importance to cholesterol homeostasis was intuitively appreciated since it was known that 50% of cholesterol degradation occurred via its catabolism to bile acids [4], which in turn are necessary for intestinal lipid absorption. The understanding of genetic control of the intracellular and extracellular regulation of cholesterol levels had to be discovered [5], before the role of the enterohepatic circulation of bile salts for cholesterol homeostasis could more fully be realized [1,6,7]. It was realized early on that ileectomy, either by the surgical removal of the distal ileum [8] or functionally as a result of medical disease (Crohn’s ileitis), could result in malabsorption of fat from the lack of adequate bile salt ileal reabsorption. In fact, by creating fat malabsorption, ileectomy or ileal bypass became the first successful treatments for excessive obesity. A specialized ileal bile salt transport mechanism is necessary to maintain a normal bile salt pool to ensure adequate concentration of bile salt micelles in the
0002-9610/03/$ – see front matter © 2003 Excerpta Medica Inc. All rights reserved. doi:10.1016/S0002-9610(02)01212-6
R.N. Redinger / The American Journal of Surgery 185 (2003) 168 –172
intestine thereby preventing fat malabsorption [9]. Furthermore, bile salt secretory diarrhea results from the effects of excessive bile salt loss into the colon consequent to their effects upon cyclic adenosine monophosphate (cAMP) induced colonic water secretion in the colon [10]. It was found that greater losses of bile salts from surgical ileectomy lowered serum cholesterol by accelerating hepatic catabolism of cholesterol to bile acids [11]. However, better understanding of bile acid synthesis was complicated by relatively large numbers of intermediatary enzymatic steps and lack of clinical relevance for different bile acid species [12]. An early clue to the differential effects of specific bile salts on membrane function and lipid solubility was realized when ursodeoxycholic acid, the epimer of the primary bile acid, chenodeoxycholic acid, was found to be superior to chenodeoxycholic acid (and other more hydrophobic bile acids) in its ability to improve bile acid induced liver cholestasis [2]. Cholic acids and chenodeoxycholic acids are the human’s major primary bile acids. All bile acids including the secondary bile acids deoxycholic and lithocholic acids, which are formed by intestinal bacteria 7␣ dehydroxylation of their respective primary bile salts, are avidly reabsorbed by an active bile salt reabsorptive mechanism in the terminal ileum. Thus, less than 5% of the bile salt pool is lost daily in feces requiring only that amount of newly synthesized bile acids by the liver to maintain the bile salt pool (Fig. 1) [11]. The full significance of new knowledge relating to the identification of the alternate or acidic pathway of hepatic bile acid synthesis favoring chenodeoxycholic acid synthesis rather than that of cholic acid [6] and the variable effects of different bile acids on hepatic cholestasis [13] required a better understanding of the molecular biology of bile acids relating to cholesterol homeostasis [14]. The profound effect of perturbed bile salt molecular biology was still not appreciated for dyslipidemia [14] that cause tissue deposition of cholesterol esters involved in the pathophysiology of atherosclerosis. Likewise, even the excessive secretion of hepatic cholesterol, or conversely the lack of adequate bile acids in bile while being the key to increased cholesterol saturation and important in the pathogeneous of cholesterol gallstones, were not appreciated for their effects on cholesterol homeostasis. But why the need for an enterohepatic circulation of bile salts in mammals? Eukaryotic cells required the incorporation of rigid amphipathic molecules such as cholesterol and phospholipids into their multiple membrane cellular organelles to accomplish adequate membrane functions [15]. However, these cells could not degrade the cholesterol steroid ring required as a result of membrane turnover but required selective P450 enzymes to modify cholesterol for its catabolism. Such enzyme activity results in multiple hydroxylation steps and side chain shortening of the carbon 27 cholesterol molecule to form carbon 24 bile acids [12,16]. The latter, smaller, amphipathic detergent-like molecules could then solubilize both cholesterol and phospho-
169
Fig. 1. The enterohepatic circulation of bile salts (BS) is illustrated showing daily hepatic bile acid synthesis matching daily fecal bile salt loss. Physiological correlation of feed-forward secretion of bile salts is shown resulting from activation of cholesterol 7␣ hydroxylase which catabolizes cholesterol to bile acids. Conversely, feedback control of bile acid synthesis by bile salts returning via the portal vein to the liver acts to repress cholesterol 7␣ hydroxylase enzyme activity.
lipids in bile forming mixed micelles to allow the transport of otherwise insoluble cholesterol from the liver for delivery by bile ducts into the intestinal tract for absorption [3,17]. Furthermore, bile salts also solubilize products of intraluminal intestinal lipid digestion [4] such as monoglycerides, fatty acids, and fat-soluble vitamins (A, D, E and K) as well as biliary cholesterol for upper intestinal lipid absorption. Thereafter, reassembly of these lipids with apoproteins within the intestinal mucosa or the liver to form lipoproteins allows aqueous transport in blood for widespread distribution of lipid to the body in analogous fashion to the role of bile salt micelles in bile for intestinal lipid absorption. A constant bile salt pool, however, needs to be maintained for effective lipid absorption and be conserved by constant reabsorption from the ileum during repeated bile salt recycling between the liver and intestine for its enterohepatic circulation [9,11]. The importance of the enterohepatic circulation of bile salts for major molecular actions required greater understanding of concomitant feedback and feed-forward regulation of major enzymatic steps involved in the control of both cholesterol and bile acid metabolism [15]. First, and foremost, was the discovery of sterol regulatory element binding proteins (SREBP) which, as members of the family of
170
R.N. Redinger / The American Journal of Surgery 185 (2003) 168 –172
membrane-bound transcription factors, encode enzymes that mediate the repression of HMG CoA reductase, the rate limiting enzyme involved in cholesterol synthesis [5,18]. The concept that intracellular cholesterol levels were controlled by feedback control of cholesterol synthesis was clarified by finding that cholesterol itself acted as a ligand for membrane-bound receptor sensors that would signal a sequence of genetic activity to effect feedback control of cholesterol synthesis. The molecular sequence involved the release of transcription factors to activate genes encoding end-product activation of key enzyme activity involved in cholesterol synthesis [19]. Thus intracellular cholesterol, when deficient for cellular needs, act as ligands to signal specific receptor molecular sensors to activate transcription factors encoding the key enzymes that would activate cholesterol synthesis within cells [19,20]. In eukaryotes, many nutrients besides cholesterol are well known to affect their metabolism through regulation of feedback control of gene expression [15]. Molecular signals, either from ligands such as cholesterol metabolites, called oxysterols, or bile salts in tissues within the territory of the enterohepatic circulation, have now been found to regulate both cholesterol and bile acid synthesis [1,15,21]. The action of bile salts as ligands for nuclear hormone receptors, which signal feedback control of bile acid synthesis, works in tandem with feed-forward regulation signaled by oxysterol ligands for cholesterol catabolism to bile salts. These findings built upon the earlier discoveries in the laboratories of Brown and Goldstein [5], who unraveled the understanding of molecular mechanisms involved in feedback control of cholesterol synthesis [18], now compliments the understanding of feed-forward control of cholesterol catabolism to bile salts and brings new meanings to the enterohepatic circulation of bile salts [1,15,21]. What was required was to discover similar molecular signaling pathways within the bile salt enterohepatic circulation that established feed-forward control of bile salt metabolism, and be complimentary to the regulation of cholesterol homeostasis by controlling its catabolism to bile acids. Nuclear bound transcription factors activated by nuclear hormone receptors had previously been established as master regulators of cholesterol homeostasis including that of its feedback regulation [6]. Receptors that had been identified without known ligands that might signal as yet unknown molecular pathways were called orphan nuclear receptors. A novel strategy of reverse endocrinology was then invoked to identify previously unknown signaling pathways by testing potential ligands for these orphan nuclear receptors [15]. By discovering that bile salts were the ligands for select members of the family of previous orphan nuclear hormone receptors, ie, those receptors previously without known ligands, a number of laboratories were able to discover the key regulatory steps by which bile salt metabolism controlled cholesterol elimination and catabolism [1,6]. What has now become apparent is that there are a num-
Fig. 2. Example of nuclear hormone receptor LXR/RXR, which is a heterodimer requiring oxysterol ligand binding for molecular signaling. A response element with specific DNA motif is required for transcription factor (TF) activation of the enzyme, CYP7A1, for feed-forward activation of cholesterol 7␣ hydroxylase.
ber of orphan nuclear hormone receptors in tissues within the territory of enterohepatic circulation of bile salts that are involved as sensors that regulate both cellular cholesterol and bile salt levels in these tissues [7]. It was also found that oxidized metabolites of cholesterol called oxysterols, were involved in signaling feed-forward metabolic pathways involved in cholesterol homeostasis [20]. Oxysterols are therefore steroid signaling molecules or ligands, which after binding to nuclear receptors, modulate transcription factors to control excessive intracellular cholesterol accumulation by regulating cholesterol catabolism to bile acids [17]. Thus both bile salts and oxysterols were identified as ligands for receptors in the signaling pathways whereby a ligand would bind to a nuclear hormone receptor of special construct (ie, dimerized to a basic retinoid receptor). These receptors would then signal a DNA response element, and sequentially activate specific transcription factors to encode activation or repression of key enzyme steps for the regulation of either bile acids or cholesterol synthesis, respectively. Thus, liver X receptor (LXR), originally found in the liver, is dimerized to another nuclear hormone receptor, retinoid X receptor ([RXR] see Fig. 2). This complex is the key receptor (ie, LXR/RXR) capable of signalling feed-forward genetic control of cholesterol homeostasis [22]. This receptor recognizes ligands termed oxysterols, which are early metabolites of cholesterol found first in diverse tissue such as on liver, brain, and gonads, that are ligands for the liver X receptor (LXR/RXR dimer) also found in those tissues [1,15,22]. Through a series of nuclear events, the up-regulation of the enzyme CYP7A1 (cholesterol 7␣ hydroxylase), a member of the family of P450 enzymes [23], activates bile acid synthesis [15]. This key receptor (LXR/RXR), once signaled by its ligand, works to initiate a molecular pathway to catabolize cholesterol to bile acids thereby lowering intracellular cholesterol levels by invoking feed-forward control of cholesterol conversion to bile acid synthesis. Another orphan nuclear receptor, farnesoid X receptor (FXR), was deorphanized once bile salts were found to be its ligand [24] and operates in an opposing fashion to that of LXR to repress bile acid synthesis in tissues within the enterohepatic circulation of bile salts [22]. With bile salts as its ligand, FXR/RXR acts as a sensor of intracellular bile
R.N. Redinger / The American Journal of Surgery 185 (2003) 168 –172
salt levels, and by similar dimerization to RXR, signals DNA element binding and induction of the small heterodimer partner (SHP) to express transcription factor activation to repress cholesterol 7␣ hydroxylation (CYP7A1) [21]. Thus, there is down-regulation of CYP7A1 enzyme activity to repress bile acid synthesis by feedback control from bile salts returning to the liver from the intestine. This action is needed to prevent intracellular toxicity from excessive bile salt levels. The FXR/RXR receptor, upon bile salt ligand binding, also acts to invoke control of bile acid synthesis to accommodate the need for a constant bile salt pool size within the enterohepatic circulation. Thus, only those bile salts lost into the colon for fecal elimination are replaced by new bile acid synthesis, which is genetically controlled to maintain bile salt homeostasis, eg, the bile salt pool size. However, what was perplexing to researchers was the need to find other orphan nuclear receptors to clarify the complete picture of molecular sensing for feedback enzymatic regulation. The bile salt sensor, FXR/RXR, causes transcription of another nuclear hormone receptor, the SHP [25], which by inhibiting nuclear receptor homolog 1, eg, LRH-1 [21,25], the competence factor for FXR/RXR, indirectly represses CYP7A1. Thus, feed-forward control of cholesterol catabolism to bile acid and feedback control of bile acid synthesis itself are regulated by coordinated actions of multiple nuclear hormone receptors within the enterohepatic circulation to maintain a regular bile salt pool size and tolerable intracellular hepatocyte bile salt concentrations [1]. An incidental benefit of such regulation is to protect the hepatocyte from bile salt mediated damage, which would create cholestatic injury if intracellular bile salt concentrations rose to toxic levels [13]. The intricacies of nuclear hormone receptor function for regulation of other gene-related protein products within the enterohepatic circulation are even more profound. It has also recently been found that the dimerized bile acid receptor (FXR/RXR) also enhances reabsorption of bile salts in the distal small bowel by enhancing activity of the ileal bile acid binding protein transporter (IBABP) [26] to resorb ileal bile salts thereby assisting to preserve the bile salt pool. It also regulates phospholipid transfer protein activity [27] and bile salt transport out of the hepatocyte at the canalicular level by activating the bile salt export pump (BSEP) [28]. Additionally FXR/RXR, through bile salt ligand binding, down-regulates the sodium taurocholate polypeptide transporter (NTCP) at the basolateral membrane to protect the hepatocyte from excessive bile acid uptake. Thus, while feed-forward and feedback control of bile salt metabolism are coordinated by nuclear receptors, they also regulate bile salt transport at hepatic and intestinal levels to fine-tune the preservation of the enterohepatic circulation of bile salts and protect the hepatocyte from excessive intracellular bile acid concentration. Finally, very recent findings indicate that the activity of LXR/RXR does more than function to enhance
171
cholesterol catabolism by CYP7A1 enzyme activity in the liver. It also increases cholesterol efflux from extrahepatic cells [29,30] by enhancing the dedicated ABC-1 cassette transporter system of these cells for cholesterol efflux from them and allow enhanced uptake of this cellularly derived cholesterol by HDL uptake and reverse cholesterol transport back to the liver for further metabolism [15,30]. In summary, through molecular signaling, the appropriate ligands, oxysterol and bile salts, bind to their respective nuclear hormone receptor sensors so that genetic transcription is accomplished to either enhance or repress cholesterol or bile acid synthesis, or both, thereby achieving normal cholesterol and bile salt homeostasis, respectively. Simultaneously they enhance transporters to control intrahepatic bile salt levels while regulating the size of the bile salt pool for its enterohepatic circulation. They also enhance cholesterol efflux from cells for transport back to the liver for further catabolism. All nuclear hormone receptors function in a coordinated fashion at hepatocyte, ileal, and peripheral tissue level, so as to achieve normal cholesterol and bile salt intracellular levels. Bile salts as ligands for nuclear hormone receptors function to regulate cholesterol homeostasis in a major way, which is in addition to their lipid detergent function for biliary cholesterol and intestinal lipid absorption. Thus, it is very likely that genetic disorders of cholesterol metabolism are linked to abnormalities of nuclear hormone receptors. Pharmaceutical manipulations, therefore, of these receptors by the use of agonists or antagonists may now be found and tested in clinical trials to correct cholesterol and/or bile salt perturbed metabolism that has contributed to excessive cholesterol deposition and atherosclerosis. Surgical ileectomy provided great insight into essential elements of cholesterol and bile acid homeostasis in that by interfering with bile salt reabsorption it enhanced cholesterol catabolism to bile acids. The underlying molecular controls initiated by multiple nuclear hormone receptors now promise further interventions that may help combat excessive cholesterol deposition in the vascular endothelium that contributes to atherosclerosis. Similarly, better treatment of intrahepatic cholestatic disorders may be found by modulating the regulation of transport proteins for bile salt metabolism.
References [1] Repa JJ, Mangelsdorf DJ. Nuclear receptor regulation of cholesterol and bile acid metabolism. Curr Opin Biotechnol 1999;10:557– 63. [2] Carey MC. The enterohepatic circulation. In: Arias I, Popper H, et al, editors. The liver: biology and pathobiology. New York: Raven Press, 1982, p 429 – 65. [3] Hoffman AF. Bile acids. In: Arias IM, Buyer JL, et al, editors. Biology and pathophysiology. New York: Raven Press, 1994, p 677–717. [4] Hoffman AF. Intestinal absorption of bile acids and biliary constituents: the intestinal components of the enterohepatic circulation and the integrated system. In: Johnson LR, editor. Physiology of the
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R.N. Redinger / The American Journal of Surgery 185 (2003) 168 –172 gastrointestinal tract. New York: Raven Press, New York, 1994, p 1845– 65. Brown MS, Goldstein JL. A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood. Proc Natl Acad Sci USA 1999;96:11041– 8. Repa JJ, Mangelsdorf DJ. The role of orphan nuclear receptors in the regulation of cholesterol homeostasis. Annu Rev Cell Dev Biol 2000; 16:459 – 81. Makishima M, Okamoto AY, Repa JJ, et al. Identification of a nuclear receptor for bile acids. Science 1999;284:1362–5. Kern F. Bile salt malabsorption in regional ileitis, ileal resection and mannitol induced diarrhea. J Clin Invest 1968;47:261. Hoffman AF. Enterohepatic circulation of bile acids. In: Shultz SG, Forte JG, Rainer BB, editors. Handbook of physiology. Vol 3. New York: Oxford University Press, 1989, p 567–96. Thaysen EH, Pedersen L. Idiopathic bile acid catharsis. Gut 1976;17: 965–70. Small DM, Dowling RH, Redinger RN. The enterohepatic circulation of bile salts. Arch Intern Med 1972;130:552–73. Russell DW, Setchell KD. Bile acid biosynthesis. Biochemistry 1992; 31:4737– 49. Xie W, Radominska-Pandya A, Shi Y, et al. An essential role for nuclear receptors SXR/PXR in detoxification of cholestatic bile acids. Proc Natl Acad Sci USA 2001;98:3375– 80. Sinal CJ, Tohkin M, Miyata M, et al. Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis. Cell 2000;102:731– 44. Schoonjans K, Brendel C, Mangelsdorf D, Auwerx J. Sterols and gene expression: control of affluence. Biochim Biophys Acta 2000; 1529:114 –25. Lecureur V, Courtois A, Payen L, et al. Expression and regulation of hepatic drug and bile acid transporters. Toxicology 2000;153:203–19. Hofmann AF, Small DM. Detergent properties of bile salts: correlation with physiological function. Annu Rev Med 1967;18:333–76. Wang X, Sato R, Brown MS, et al. SREBP-1, a membrane-bound transcription factor released by sterol-regulated proteolysis. Cell 1994;77:53– 62.
[19] Vallett SM, Sanchez HB, Rosenfeld JM, Osborne TF. A direct role for sterol regulatory element binding protein in activation of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene. J Biol Chem 1996;271:12247–53. [20] Russell DW. Oxysterol biosynthetic enzymes. Biochim Biophys Acta 2000;1529:126 –35. [21] Lu TT, Makishima M, Repa JJ, et al. Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors. Mol Cell 2000;6:507–15. [22] Willy PJ, Umesono K, Ong ES, et al. LXR, a nuclear receptor that defines a distinct retinoid response pathway. Genes Dev 1995;9: 1033– 45. [23] Waxman DJ. P450 gene induction by structurally diverse xenochemicals: central role of nuclear receptors CAR, PXR, and PPAR. Arch Biochem Biophys 1999;369:11–23. [24] Tu H, Okamoto AY, Shan B. FXR, a bile acid receptor and biological sensor. Trends Cardiovasc Med 2000;10:30 –5. [25] Goodwin B, Jones SA, Price RR, et al. A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis. Mol Cell 2000;6:517–26. [26] Grober J, Zaghini I, Fujii H, et al. Identification of a bile acidresponsive element in the human ileal bile acid-binding protein gene. Involvement of the farnesoid X receptor/9-cis-retinoic acid receptor heterodimer. J Biol Chem 1999;274:29749 –54. [27] Urizar NL, Dowhan DH, Moore DD. The farnesoid X-activated receptor mediates bile acid activation of phospholipid transfer protein gene expression. J Biol Chem 2000;275:39313–7. [28] Ananthanarayanan M, Balasubramanian N, Makishima M, et al. Human bile salt export pump promoter is transactivated by the farnesoid X receptor/bile acid receptor. J Biol Chem 2001;276:28857– 65. [29] Repa JJ, Turley SD, Lobaccaro JA, et al. Regulation of absorption and ABC1-mediated efflux of cholesterol by RXR heterodimers. Science 2000;289:1524 –9. [30] Drobnik W, Lindenthal B, Lieser B, et al. ATP-binding cassette transporter A1 (ABCA1) affects total body sterol metabolism. Gastroenterology 2001;120:1203–11.
Review
The coming of age of our understanding of the enterohepatic circulation of bile salts Richard N. Redinger, M.D.* Department of Medicine, University of Louisville, 530 South Jackson St., 3rd Floor, Louisville, KY 40292, USA Manuscript received April 22, 2002; revised manuscript August 12, 2002
Abstract Recent advances in molecular biology have greatly accelerated knowledge relating to the significance of the enterohepatic circulation of bile salts. This review highlights the role that both oxysterols and bile salts play as ligands which, when bound to nuclear hormone receptors, activate transcription factors that set into play feed-forward catabolism of cholesterol to bile salts and feedback control of bile acid synthesis. The nuclear hormone receptors, liver X receptor (LXR) and farnesoid X receptor (FXR) both combined as heterodimers with retinoid X receptor and with oxysterols and bile salts, respectively as their ligands, initiate powerful genetic controls over cholesterol and bile acid homeostatic mechanisms. LXR/RXR signals molecular control of feed-forward catabolism of cholesterol to bile acids while FXR/RXR initiates feedback control of bile acid synthesis. An additional nuclear hormone receptor, small heterodimer partner (SHP), is required to inhibit the competence factor, liver receptor homolog-1 to achieve repression of bile acid synthesis in the liver and in so doing SHP autoregulates its own function. Additionally, while bile acid synthesis is repressed, pool size is preserved by the action of FXR/RXR at both hepatic and intestinal levels, which genetically signals enhanced hepatocyte bile salt transport by the bile salt export pump (BSEP) and the ileal bile acid binding protein (IBABP) for ileal reabsorption. During activation of cholesterol catabolism, LXR/RXR enhances reverse cholesterol transport by increasing cholesterol efflux via the ABC-1 transporter from extrahepatic cells. This cholesterol is then taken up by high-density lipoprotein (HDL) and transported back to the liver for further cholesterol catabolism and elimination in bile. The genetic coordination of nuclear hormone receptor function within the territory of the enterohepatic of bile salts allows for normal cholesterol and bile acid homeostasis thereby preventing atherosclerosis. © 2003 Excerpta Medica Inc. All rights reserved. Keywords: Nuclear hormone receptors; Bile salt; Cholesterol homeostasis
Recent advances in molecular biology have greatly accelerated knowledge relating to the significance of the enterohepatic circulation of bile salts [1]. (Bile acids are synthesized with a terminal acidic group, but when conjugated with glycine or taurine and at physiological pH, they are negatively charged and exist in their anion form. In this review, the term “bile acid” denotes synthesis whereas as “bile salts” denotes their physiological state at normal cellular pH.) The great hope that bile salts would provide an effective medical treatment for cholesterol gallstone disease had not fulfilled enthusiastic expectations [2] and with the widespread practice of safe laparoscopic cholecystectomy, bile acid research had languished for almost two decades awaiting further advances in molecular biology. The clinical significance of preserving the bile salt pool for lipid absorp-
* Corresponding author. Tel.: ⫹1-502-852-5241; fax: ⫹1-502-8526233.
tion [3] and its importance to cholesterol homeostasis was intuitively appreciated since it was known that 50% of cholesterol degradation occurred via its catabolism to bile acids [4], which in turn are necessary for intestinal lipid absorption. The understanding of genetic control of the intracellular and extracellular regulation of cholesterol levels had to be discovered [5], before the role of the enterohepatic circulation of bile salts for cholesterol homeostasis could more fully be realized [1,6,7]. It was realized early on that ileectomy, either by the surgical removal of the distal ileum [8] or functionally as a result of medical disease (Crohn’s ileitis), could result in malabsorption of fat from the lack of adequate bile salt ileal reabsorption. In fact, by creating fat malabsorption, ileectomy or ileal bypass became the first successful treatments for excessive obesity. A specialized ileal bile salt transport mechanism is necessary to maintain a normal bile salt pool to ensure adequate concentration of bile salt micelles in the
0002-9610/03/$ – see front matter © 2003 Excerpta Medica Inc. All rights reserved. doi:10.1016/S0002-9610(02)01212-6
R.N. Redinger / The American Journal of Surgery 185 (2003) 168 –172
intestine thereby preventing fat malabsorption [9]. Furthermore, bile salt secretory diarrhea results from the effects of excessive bile salt loss into the colon consequent to their effects upon cyclic adenosine monophosphate (cAMP) induced colonic water secretion in the colon [10]. It was found that greater losses of bile salts from surgical ileectomy lowered serum cholesterol by accelerating hepatic catabolism of cholesterol to bile acids [11]. However, better understanding of bile acid synthesis was complicated by relatively large numbers of intermediatary enzymatic steps and lack of clinical relevance for different bile acid species [12]. An early clue to the differential effects of specific bile salts on membrane function and lipid solubility was realized when ursodeoxycholic acid, the epimer of the primary bile acid, chenodeoxycholic acid, was found to be superior to chenodeoxycholic acid (and other more hydrophobic bile acids) in its ability to improve bile acid induced liver cholestasis [2]. Cholic acids and chenodeoxycholic acids are the human’s major primary bile acids. All bile acids including the secondary bile acids deoxycholic and lithocholic acids, which are formed by intestinal bacteria 7␣ dehydroxylation of their respective primary bile salts, are avidly reabsorbed by an active bile salt reabsorptive mechanism in the terminal ileum. Thus, less than 5% of the bile salt pool is lost daily in feces requiring only that amount of newly synthesized bile acids by the liver to maintain the bile salt pool (Fig. 1) [11]. The full significance of new knowledge relating to the identification of the alternate or acidic pathway of hepatic bile acid synthesis favoring chenodeoxycholic acid synthesis rather than that of cholic acid [6] and the variable effects of different bile acids on hepatic cholestasis [13] required a better understanding of the molecular biology of bile acids relating to cholesterol homeostasis [14]. The profound effect of perturbed bile salt molecular biology was still not appreciated for dyslipidemia [14] that cause tissue deposition of cholesterol esters involved in the pathophysiology of atherosclerosis. Likewise, even the excessive secretion of hepatic cholesterol, or conversely the lack of adequate bile acids in bile while being the key to increased cholesterol saturation and important in the pathogeneous of cholesterol gallstones, were not appreciated for their effects on cholesterol homeostasis. But why the need for an enterohepatic circulation of bile salts in mammals? Eukaryotic cells required the incorporation of rigid amphipathic molecules such as cholesterol and phospholipids into their multiple membrane cellular organelles to accomplish adequate membrane functions [15]. However, these cells could not degrade the cholesterol steroid ring required as a result of membrane turnover but required selective P450 enzymes to modify cholesterol for its catabolism. Such enzyme activity results in multiple hydroxylation steps and side chain shortening of the carbon 27 cholesterol molecule to form carbon 24 bile acids [12,16]. The latter, smaller, amphipathic detergent-like molecules could then solubilize both cholesterol and phospho-
169
Fig. 1. The enterohepatic circulation of bile salts (BS) is illustrated showing daily hepatic bile acid synthesis matching daily fecal bile salt loss. Physiological correlation of feed-forward secretion of bile salts is shown resulting from activation of cholesterol 7␣ hydroxylase which catabolizes cholesterol to bile acids. Conversely, feedback control of bile acid synthesis by bile salts returning via the portal vein to the liver acts to repress cholesterol 7␣ hydroxylase enzyme activity.
lipids in bile forming mixed micelles to allow the transport of otherwise insoluble cholesterol from the liver for delivery by bile ducts into the intestinal tract for absorption [3,17]. Furthermore, bile salts also solubilize products of intraluminal intestinal lipid digestion [4] such as monoglycerides, fatty acids, and fat-soluble vitamins (A, D, E and K) as well as biliary cholesterol for upper intestinal lipid absorption. Thereafter, reassembly of these lipids with apoproteins within the intestinal mucosa or the liver to form lipoproteins allows aqueous transport in blood for widespread distribution of lipid to the body in analogous fashion to the role of bile salt micelles in bile for intestinal lipid absorption. A constant bile salt pool, however, needs to be maintained for effective lipid absorption and be conserved by constant reabsorption from the ileum during repeated bile salt recycling between the liver and intestine for its enterohepatic circulation [9,11]. The importance of the enterohepatic circulation of bile salts for major molecular actions required greater understanding of concomitant feedback and feed-forward regulation of major enzymatic steps involved in the control of both cholesterol and bile acid metabolism [15]. First, and foremost, was the discovery of sterol regulatory element binding proteins (SREBP) which, as members of the family of
170
R.N. Redinger / The American Journal of Surgery 185 (2003) 168 –172
membrane-bound transcription factors, encode enzymes that mediate the repression of HMG CoA reductase, the rate limiting enzyme involved in cholesterol synthesis [5,18]. The concept that intracellular cholesterol levels were controlled by feedback control of cholesterol synthesis was clarified by finding that cholesterol itself acted as a ligand for membrane-bound receptor sensors that would signal a sequence of genetic activity to effect feedback control of cholesterol synthesis. The molecular sequence involved the release of transcription factors to activate genes encoding end-product activation of key enzyme activity involved in cholesterol synthesis [19]. Thus intracellular cholesterol, when deficient for cellular needs, act as ligands to signal specific receptor molecular sensors to activate transcription factors encoding the key enzymes that would activate cholesterol synthesis within cells [19,20]. In eukaryotes, many nutrients besides cholesterol are well known to affect their metabolism through regulation of feedback control of gene expression [15]. Molecular signals, either from ligands such as cholesterol metabolites, called oxysterols, or bile salts in tissues within the territory of the enterohepatic circulation, have now been found to regulate both cholesterol and bile acid synthesis [1,15,21]. The action of bile salts as ligands for nuclear hormone receptors, which signal feedback control of bile acid synthesis, works in tandem with feed-forward regulation signaled by oxysterol ligands for cholesterol catabolism to bile salts. These findings built upon the earlier discoveries in the laboratories of Brown and Goldstein [5], who unraveled the understanding of molecular mechanisms involved in feedback control of cholesterol synthesis [18], now compliments the understanding of feed-forward control of cholesterol catabolism to bile salts and brings new meanings to the enterohepatic circulation of bile salts [1,15,21]. What was required was to discover similar molecular signaling pathways within the bile salt enterohepatic circulation that established feed-forward control of bile salt metabolism, and be complimentary to the regulation of cholesterol homeostasis by controlling its catabolism to bile acids. Nuclear bound transcription factors activated by nuclear hormone receptors had previously been established as master regulators of cholesterol homeostasis including that of its feedback regulation [6]. Receptors that had been identified without known ligands that might signal as yet unknown molecular pathways were called orphan nuclear receptors. A novel strategy of reverse endocrinology was then invoked to identify previously unknown signaling pathways by testing potential ligands for these orphan nuclear receptors [15]. By discovering that bile salts were the ligands for select members of the family of previous orphan nuclear hormone receptors, ie, those receptors previously without known ligands, a number of laboratories were able to discover the key regulatory steps by which bile salt metabolism controlled cholesterol elimination and catabolism [1,6]. What has now become apparent is that there are a num-
Fig. 2. Example of nuclear hormone receptor LXR/RXR, which is a heterodimer requiring oxysterol ligand binding for molecular signaling. A response element with specific DNA motif is required for transcription factor (TF) activation of the enzyme, CYP7A1, for feed-forward activation of cholesterol 7␣ hydroxylase.
ber of orphan nuclear hormone receptors in tissues within the territory of enterohepatic circulation of bile salts that are involved as sensors that regulate both cellular cholesterol and bile salt levels in these tissues [7]. It was also found that oxidized metabolites of cholesterol called oxysterols, were involved in signaling feed-forward metabolic pathways involved in cholesterol homeostasis [20]. Oxysterols are therefore steroid signaling molecules or ligands, which after binding to nuclear receptors, modulate transcription factors to control excessive intracellular cholesterol accumulation by regulating cholesterol catabolism to bile acids [17]. Thus both bile salts and oxysterols were identified as ligands for receptors in the signaling pathways whereby a ligand would bind to a nuclear hormone receptor of special construct (ie, dimerized to a basic retinoid receptor). These receptors would then signal a DNA response element, and sequentially activate specific transcription factors to encode activation or repression of key enzyme steps for the regulation of either bile acids or cholesterol synthesis, respectively. Thus, liver X receptor (LXR), originally found in the liver, is dimerized to another nuclear hormone receptor, retinoid X receptor ([RXR] see Fig. 2). This complex is the key receptor (ie, LXR/RXR) capable of signalling feed-forward genetic control of cholesterol homeostasis [22]. This receptor recognizes ligands termed oxysterols, which are early metabolites of cholesterol found first in diverse tissue such as on liver, brain, and gonads, that are ligands for the liver X receptor (LXR/RXR dimer) also found in those tissues [1,15,22]. Through a series of nuclear events, the up-regulation of the enzyme CYP7A1 (cholesterol 7␣ hydroxylase), a member of the family of P450 enzymes [23], activates bile acid synthesis [15]. This key receptor (LXR/RXR), once signaled by its ligand, works to initiate a molecular pathway to catabolize cholesterol to bile acids thereby lowering intracellular cholesterol levels by invoking feed-forward control of cholesterol conversion to bile acid synthesis. Another orphan nuclear receptor, farnesoid X receptor (FXR), was deorphanized once bile salts were found to be its ligand [24] and operates in an opposing fashion to that of LXR to repress bile acid synthesis in tissues within the enterohepatic circulation of bile salts [22]. With bile salts as its ligand, FXR/RXR acts as a sensor of intracellular bile
R.N. Redinger / The American Journal of Surgery 185 (2003) 168 –172
salt levels, and by similar dimerization to RXR, signals DNA element binding and induction of the small heterodimer partner (SHP) to express transcription factor activation to repress cholesterol 7␣ hydroxylation (CYP7A1) [21]. Thus, there is down-regulation of CYP7A1 enzyme activity to repress bile acid synthesis by feedback control from bile salts returning to the liver from the intestine. This action is needed to prevent intracellular toxicity from excessive bile salt levels. The FXR/RXR receptor, upon bile salt ligand binding, also acts to invoke control of bile acid synthesis to accommodate the need for a constant bile salt pool size within the enterohepatic circulation. Thus, only those bile salts lost into the colon for fecal elimination are replaced by new bile acid synthesis, which is genetically controlled to maintain bile salt homeostasis, eg, the bile salt pool size. However, what was perplexing to researchers was the need to find other orphan nuclear receptors to clarify the complete picture of molecular sensing for feedback enzymatic regulation. The bile salt sensor, FXR/RXR, causes transcription of another nuclear hormone receptor, the SHP [25], which by inhibiting nuclear receptor homolog 1, eg, LRH-1 [21,25], the competence factor for FXR/RXR, indirectly represses CYP7A1. Thus, feed-forward control of cholesterol catabolism to bile acid and feedback control of bile acid synthesis itself are regulated by coordinated actions of multiple nuclear hormone receptors within the enterohepatic circulation to maintain a regular bile salt pool size and tolerable intracellular hepatocyte bile salt concentrations [1]. An incidental benefit of such regulation is to protect the hepatocyte from bile salt mediated damage, which would create cholestatic injury if intracellular bile salt concentrations rose to toxic levels [13]. The intricacies of nuclear hormone receptor function for regulation of other gene-related protein products within the enterohepatic circulation are even more profound. It has also recently been found that the dimerized bile acid receptor (FXR/RXR) also enhances reabsorption of bile salts in the distal small bowel by enhancing activity of the ileal bile acid binding protein transporter (IBABP) [26] to resorb ileal bile salts thereby assisting to preserve the bile salt pool. It also regulates phospholipid transfer protein activity [27] and bile salt transport out of the hepatocyte at the canalicular level by activating the bile salt export pump (BSEP) [28]. Additionally FXR/RXR, through bile salt ligand binding, down-regulates the sodium taurocholate polypeptide transporter (NTCP) at the basolateral membrane to protect the hepatocyte from excessive bile acid uptake. Thus, while feed-forward and feedback control of bile salt metabolism are coordinated by nuclear receptors, they also regulate bile salt transport at hepatic and intestinal levels to fine-tune the preservation of the enterohepatic circulation of bile salts and protect the hepatocyte from excessive intracellular bile acid concentration. Finally, very recent findings indicate that the activity of LXR/RXR does more than function to enhance
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cholesterol catabolism by CYP7A1 enzyme activity in the liver. It also increases cholesterol efflux from extrahepatic cells [29,30] by enhancing the dedicated ABC-1 cassette transporter system of these cells for cholesterol efflux from them and allow enhanced uptake of this cellularly derived cholesterol by HDL uptake and reverse cholesterol transport back to the liver for further metabolism [15,30]. In summary, through molecular signaling, the appropriate ligands, oxysterol and bile salts, bind to their respective nuclear hormone receptor sensors so that genetic transcription is accomplished to either enhance or repress cholesterol or bile acid synthesis, or both, thereby achieving normal cholesterol and bile salt homeostasis, respectively. Simultaneously they enhance transporters to control intrahepatic bile salt levels while regulating the size of the bile salt pool for its enterohepatic circulation. They also enhance cholesterol efflux from cells for transport back to the liver for further catabolism. All nuclear hormone receptors function in a coordinated fashion at hepatocyte, ileal, and peripheral tissue level, so as to achieve normal cholesterol and bile salt intracellular levels. Bile salts as ligands for nuclear hormone receptors function to regulate cholesterol homeostasis in a major way, which is in addition to their lipid detergent function for biliary cholesterol and intestinal lipid absorption. Thus, it is very likely that genetic disorders of cholesterol metabolism are linked to abnormalities of nuclear hormone receptors. Pharmaceutical manipulations, therefore, of these receptors by the use of agonists or antagonists may now be found and tested in clinical trials to correct cholesterol and/or bile salt perturbed metabolism that has contributed to excessive cholesterol deposition and atherosclerosis. Surgical ileectomy provided great insight into essential elements of cholesterol and bile acid homeostasis in that by interfering with bile salt reabsorption it enhanced cholesterol catabolism to bile acids. The underlying molecular controls initiated by multiple nuclear hormone receptors now promise further interventions that may help combat excessive cholesterol deposition in the vascular endothelium that contributes to atherosclerosis. Similarly, better treatment of intrahepatic cholestatic disorders may be found by modulating the regulation of transport proteins for bile salt metabolism.
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