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Intestinal and hepatic Niemann–Pick C1L1 proteins: Future therapeutic targets for cholesterol gallstones disease?
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Intestinal and hepatic Niemann-Pick C1L1 proteins: future therapeutic targets for cholesterol gallstones disease? Ibrahim Guillermo Castro-Torres, Minarda De la O-Arciniega, Gustavo Adolfo Bravo-Duarte, Janeth Gallegos-Estudillo, Miguel Ángel Domínguez-Ortíz, Mariano Martínez-Vázquez www.elsevier.com/locate/ejphar
PII: DOI: Reference:
S0014-2999(14)00103-4 http://dx.doi.org/10.1016/j.ejphar.2014.01.067 EJP69117
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European Journal of Pharmacology
Received date: 29 September 2013 Revised date: 13 January 2014 Accepted date: 24 January 2014 Cite this article as: Ibrahim Guillermo Castro-Torres, Minarda De la OArciniega, Gustavo Adolfo Bravo-Duarte, Janeth Gallegos-Estudillo, Miguel Ángel Domínguez-Ortíz, Mariano Martínez-Vázquez, Intestinal and hepatic Niemann-Pick C1L1 proteins: future therapeutic targets for cholesterol gallstones disease?, European Journal of Pharmacology, http://dx.doi.org/10.1016/j. ejphar.2014.01.067 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 Intestinal and hepatic Niemann-Pick C1L1 proteins: future therapeutic targets for cholesterol gallstones disease?
Ibrahim Guillermo Castro-Torresa*, Minarda De la O-Arciniegab, Gustavo Adolfo BravoDuartec, Janeth Gallegos-Estudillod, Miguel Ángel Domínguez-Ortízd, Mariano MartínezVázqueza
a
Instituto de Química. Universidad Nacional Autónoma de México. México, D.F.
b
Área Académica de Farmacia. Instituto de Ciencias de la Salud. Universidad Autónoma
del Estado de Hidalgo. Pachuca de Soto, Hidalgo. México. c
Facultad de Medicina “Lic. Miguel Alemán Valdés”, Universidad Veracruzana, Veracruz,
México. d
Laboratorio de Productos Naturales. Instituto de Ciencias Básicas. Universidad
Veracruzana. Xalapa de Enríquez, Veracruz. México.
Corresponding author: Ibrahim Guillermo Castro-Torres, Prof. Instituto de Química. Universidad Nacional Autónoma de México. Circuito Exterior s/n, Ciudad Universitaria. C.P. 04510. Delegación Coyoacán. México, Distrito Federal. Telephone: (+52) 55 56224403. E-mail: [email protected]
Conflicts of interest: the authors disclose no conflicts.
2 Abstract The formation of cholesterol gallstones is a very complex and polygenic disorder that involves an alteration of the secretion of bile lipids, cholesterol crystallization, important immunological reactions in the gallbladder tissue, formation of biliary sludge composed of mucin, and inadequate gallbladder motility. The search for a therapeutic target is oriented towards decreasing bile secretion and intestinal absorption of cholesterol, in which Niemann-Pick C1L1 (NPC1L1) proteins play an important role. In basic and clinical studies, regulating the expression of these proteins can reduce intestinal, liver, plasma and bile cholesterol levels, a therapeutic effect that would be useful not only for treating the disease, but to prevent it, given the large quantity of risk factors. We discuss these effects in this review and propose NPC1L1 proteins as future therapeutic targets of cholesterol gallstones disease.
Keywords: cholesterol gallstones, NPC1L1 proteins, therapeutic target, gallbladder
3 1. Introduction Cholesterol gallstones disease has many risk factors and is a global problem (Portincasa et al., 2006). Of the different types of gallstones, over 80% are formed by cholesterol; nowadays, this disease is associated with the development of atherosclerosis (MéndezSánchez et al., 2008). There are many cases of gallstones in the United States, Germany, China, Mexico, Chile and Argentina, which lead to the development of numerous gastrointestinal problems and to the performance of thousands of surgeries to remove the gallbladder (Stinton and Shaffer, 2012). Ursodeoxycholic acid is the only drug therapy for cholesterol gallstones, but it can only be used when the stones are made strictly of cholesterol and are in a gallbladder with adequate motility; otherwise, the drug may not exert its mechanism of action (Guarino et al., 2013). Another drug which has been shown to partially dissolve gallstones in humans is ezetimibe, which inhibits the NPC1L1 protein and the intestinal absorption of cholesterol (de Bari et al., 2011; Portincasa et al., 2012). Biliary excretion and intestinal absorption are important processes in the pathophysiology of lithiasis (Portincasa and Wang, 2012), but with respect to treatment, it is important to analyze the mechanisms of action that can disintegrate stones and dissolve cholesterol crystals. The NPC1L1 protein is localized in the proximal jejunum of different species and is also expressed in the liver of humans (Deffieu et al., 2011). In preclinical research, ezetimibe showed a major effect, inhibiting the formation of gallstones in mice; the mechanism of action was found in the intestine, as these rodents do not express the liver protein (Davis and Altmann, 2009). The function of the NPC1L1 protein in the liver has not been fully described yet; however, it takes part in regulating the concentration and transport of cholesterol in the intestine (Pramfalk et al., 2011, Wang and Song, 2012, Castro-Torres
4 and Bravo-Duarte, 2013), making it an essential element in the pathophysiology of biliary lithiasis.
5 2. Pathophysiology of gallstones The search for pharmacological therapies is complicated due to the complexity of the steps in which a gallstone is formed. These steps involve imbalances in the secretion of biliary cholesterol, the crystallization/nucleation of cholesterol, inflammatory reactions of the gallbladder, and the alteration of the secretion of mucin proteins; some authors also argue the importance of intestinal and gallbladder motility (Maurer et al., 2009; Castro-Torres, 2012; Krawczyk et al., 2012). When the secretion of bile lipids becomes altered, cholesterol can saturate the gallbladder and produce imbalances. In this first phase, the most important proteins are family members ATP-binding cassette transporters (ABC): ABCB11 (bile salts), ABCB4 (phospholipids), ABCG5 and ABCG8 (cholesterol transporters) (CastroTorres and Martínez-Vázquez, 2013). Regarding the transport of cholesterol, several nuclear receptors and metabolism enzymes are involved at the hepatocyte level: farnesoid receptor, liver X receptors, pregnane receptor, sterol response element binding protein SREBP-1 and SREBP-2, NPC1L1 protein, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoAR) and cholesterol 7α-hydroxylase. In addition to hepatocytes, the transport of biliary cholesterol before the lipid reaches ABCG5/8 heterodimers must be considered; in this, the high-density lipoprotein and its scavenger receptor-1 are very important (Chuang et al., 2013; Di Ciaula et al 2013). Each of these molecules deserves to be researched to look for preventive and therapeutic breakthroughs. When the vesicle has high cholesterol concentrations, the lipid may undergo crystallization, an important and limiting physiochemical change in the development of the gallstone (Abeysuriya et al 2010). Mucin proteins (5B and 5AC primarily) and other pronuclear factors are involved at this pathophysiological stage (Kim et al., 2012); here, too, there could be a therapeutic target to prevent the change of the physical state of cholesterol. The motility of the gallbladder can
6 also be studied to look for a treatment; under conditions of lithiasis, the emptying and filling of the gallbladder becomes altered, favoring the growth of stones. The hormone cholecystokinin and its main type 1 receptor, control this movement of the gallbladder (Xie et al., 2012). It has been shown that in mice susceptible to the development of lithiasis, a deficiency of the cholecystokinin receptor, promotes the development of gallstones, even after treatment with salts of ursodeoxycholic acid, the most important bile acid for dissolving cholesterol gallstones (Nihei et al., 2011). These data demonstrate the importance of the receptor in the pathophysiology of lithiasis and suggest it as a potential therapeutic target. One of the most interesting sites for gallstones therapy is the small intestine, where the absorption of cholesterol takes place. Proteins NPC1L1, ABCG5 and ABCG8 are expressed in the brush border of enterocytes, which are involved in the transport of cholesterol (Davis et al., 2011); the lipid comes mostly from the diet, so a high fat intake increases the risk of developing hypercholesterolemia and gallbladder lithiasis. Inhibiting NPC1L1 expression inhibits the intestinal absorption of cholesterol, preventing the development of gallstones and reducing plasma cholesterol levels (Tang et al., 2011); the drug ezetimibe has the ability to inhibit the expression of protein NPC1L1 and this route is of particular interest for the search for a gallstones treatment. With respect to intestinal absorption, the enzymes that allow the transport of cholesterol are very important: cholesteryl hydrolase and acyl-coenzyme A: cholesterol acyltransferase (ACAT2).The first enzyme hydrolyzes cholesterol so that it can pass through the enterocyte, and the second one esterifies it so that it can be transported in chylomicrons to the liver (Grenier et al., 2012).
7 3. Intestinal NPC1L1 protein and cholesterol transport This protein is expressed in the cells that form the brush border of the enterocytes; the region where its expression is higher is the proximal jejunum, a site of cholesterol absorption (Davis and Altmann, 2009). The human protein contains 1332 amino acids and 13 transmembrane domains, of which 5 function as sterol detection sites (Wang and Song, 2012). Dietary cholesterol is absorbed in the intestine and reaches the liver via systemic circulation; thus, the participation of the NPC1L1 protein is very important because it can determine the amount of cholesterol circulating to the liver and allow for it to be disposed as neutral sterol. Biliary cholesterol secretion may depend on intestinal absorption and hepatic cholesterol concentration. The intestine receives cholesterol mainly from food, bile secretion and, in a lesser scale, from the desquamation suffered by its epithelium (Davis et al., 2011). The transport of cholesterol through the NPC1L1 protein is highly selective, unlike what happens with other sterols; it occurs via vesicular endocytosis mediated by the protein clathrin (Wang et al., 2011). Another molecule required for intestinal cholesterol absorption, called caveolin-1, has been identified (Tang et al., 2011). Cholesterol reaches the brush border membrane of enterocyte in mixed micelles or phospholipid vesicles (liposomes); these transport complexes must release it so it can be binded by specific domains of the protein NPC1L1; if the lipid is esterified, it must be hydrolyzed by the cholesterol ester hydrolase enzyme so that it can be absorbed (Grenier et al., 2012). After cholesterol passes through the enterocyte membrane, it heads to the endoplasmic reticulum to be esterified by the ACAT2; subsequent to this, it is transported in the chylomicrons circulating in the lymphatic system of the intestine and the thoracic duct that end their route in the systemic circulation, where they reach the liver as chylomicron remnants (Castro-Torres and Martínez-Vázquez, 2013).
8 4. Basic research of intestinal NPC1L1 protein Important basic works have studied the expression of NPC1L1 protein with respect to the prevention of gallstones. Few studies focus on gallstones treatment because they tend to take into account cost-benefit considerations during the development of the disease. C57BL/6 and C57L/J mice are important models for the study of cholesterol gallstones and these strains have been used in important studies about the expression of NPC1L1 proteins in the presence of gallstones. Ezetimibe is a drug that blocks the expression of NPC1L1 and this causes a decrease in cholesterol absorption by the small intestine (Wang et al., 2013). This drug decreases the amount of cholesterol delivered to the liver and bile, thereby reducing the amount of cholesterol available for the formation of gallstones (Stein et al., 2012). An important preclinical study used male C57BL/6 mice in which gallstones were induced by a diet enriched in cholesterol and cholic acid. Mice were administered with ezetimibe at a dose of 5 mg/kg for 14 days. Ezetimibe administration reduced intestinal cholesterol absorption by 90% in control animals and by 35% in mice with cholesterol gallstones; the treatment also prevented the formation of cholesterol crystals and gallstones. Mice treated with the drug exhibited a 60% reduction in biliary cholesterol saturation index. There was a significant decrease in the expression of the NPC1L1 protein in mice fed with lithogenic diet due to the effect of ezetimibe (Zúñiga et al., 2008). The authors demonstrated that the decrease of intestinal NPC1L1 expression could be a molecular pathway for the prevention of gallstones. In another preclinical study, C57L/J mice were used to study the prevention of lithiasis. C57L/J mice fed a lithogenic diet (1% cholesterol and 0.1% cholic acid) developed gallstones in less than a month; in this model, the administration of ezetimibe at doses of 8
9 mg/kg/day reduced the expression of intestinal NPC1L1 protein. This effect produced a decrease in the intestinal absorption of cholesterol (5.0±1.4%), compared with mice afflicted with gallstones (62.5 ± 4.6%); in addition, it decreased biliary cholesterol secretion and improved gallbladder motility with no alteration of bile salt content (80.70% molar) (Wang et al., 2008). Considering that an important process of the pathophysiology of lithiasis takes place in the gallbladder, the effects caused by the inhibition of NPC1L1 may be employed in the treatment and prevention of the disease. If we speak of lithiasis prevention, decreasing intestinal cholesterol by inhibiting NPC1L1 prevents the circulation of high concentrations of the lipid towards the liver; this would also reduce cholesterol secretion in bile, preventing its accumulation in the gallbladder. With regard to treatment, when gallstones have already formed, its main component is crystallized cholesterol, but the low expression of intestinal NPC1L1 reduces bile secretion, improves motility and does not alter the content of bile salts. The latter solutes are the most abundant in the bile, and when they are in a gallbladder with adequate motility, they have the ability to emulsify the cholesterol of the gallstones, causing their partial or complete disintegration. This mechanism of action exerted by bile salts is the mechanism of ursodeoxycholic acid, but in this case, such effects are produced by the NPC1L1 protein of the small intestine, which, from a distant site to the gallbladder, controls the transport of cholesterol, preventing supersaturation in bile. A study in Golden Syrian hamsters, which were fed a high fat diet and ezetimibe at doses of 2 mg/kg/day, demonstrated that biliary cholesterol secretion decreased as a result of the pharmacological effect, which was the decrease in intestinal NPC1L1 expression. At this dose of ezetimiba, cholesterol absorption fell by 82%, fecal neutral sterol excretion increased 5.3-fold, and hepatic and intestinal cholesterol synthesis increased more than
10 twofold, but there were no significant changes in either fecal bile acid excretion or biliary lipid composition (Valasek et al., 2008). It is necessary to analyze more proteins involved in cholesterol transport and metabolism in order to find different mechanisms of action. The fact that a drug with the property of inhibiting the expression of the intestinal NPC1L1 protein already exists does not eliminate the need to research the effects that this protein can have on gallstones; it is also important to consider the liver protein, which, because of its place of expression, prevents ezetimibe from acting in the same way, and if it could do so, its effects on the concentration of cholesterol would not be the same, since the intestine and the liver have their own cell medium and their own nuclear receptors involved in the physiological regulation of the lipid.
11 5. Clinical studies of intestinal NPC1L1 protein A clinical study in 12 Chinese patients with cholesterol gallstones and 31 control patients demonstrated that, as a consequence of the disease, the mRNA levels of proteins NPC1L1 and ACAT2 (the primary enzyme responsible for esterified cholesterol) increased, resulting in an overexpression of them, whereas in the transport ABCG5/8 proteins no changes were observed. These results were obtained from protein analysis in jejunal biopsies (Jiang et al., 2009). The data indicate that biliary lithiasis produces a high concentration of intestinal cholesterol and, as the lipid circulates mostly to the liver, it must first be transported (NPC1L1 overexpression) and then esterified to enter into chylomicrons (ACAT2 overexpression); thus the downregulation of the expression of intestinal NPC1L1 could counter these cholesterol imbalances. Currently, the drug ezetimibe has the function of decreasing the intestinal absorption of cholesterol, but there is only one clinical study associated to the treatment of cholesterol gallstones, where the action of the drug (20 mg/kg/day) reduces biliary cholesterol secretion in sick patients, decreasing the size of the gallstones (Wang et al., 2008).
12 6. Hepatic NPC1L1 protein and cholesterol transport This protein is expressed in the canalicular membrane of the hepatocyte, and its role in cholesterol transport has not yet been fully explained. Within its participation in homeostasis, it regulates the expression of the Niemann-Pick C2 (NPC2) protein and involves the participation of many transcription factors (Yamanashi et al., 2011). The cholesterol linked to this protein is the one coming from the systemic circulation and that which is synthesized de novo in the liver (Temel et al., 2007). Different studies have determined that cholesterol transport throughout the hepatic NPC1L1 protein is very complex, carried out it by vesicular endocytosis mediated by a clathrin AP2 complex. The hepatic NPC1L1 protein has specific microdomains where the lipid-protein interaction can be carried out. This type of transport is carried out by lipid rafts and involves flotillin proteins 1 and 2 (Cianciola et al., 2011). The cell medium of the hepatic NPC1L1 protein and the type of genetic regulation it is subjected to, determine that its effect on cholesterol in the hepatocyte is not the same as that on intestinal cholesterol; therefore, its participation as a therapeutic target in the treatment of cholesterol gallstones must be carefully differentiated.
13 7. Basic research of hepatic NPC1L1 protein Rodents do not express the protein NPC1L1 in the liver, but it has been possible to generate transgenic models that can do it. It was determined in rat livers capable of expressing the human NPC1L1 protein, that with the increase in the expression of the liver protein, the secretion of biliary cholesterol decreases and hepatic reabsorption occurs, but it has also been shown that there is a minimal increase of cholesterol in the liver (30), indicating that cholesterol does not necessarily accumulate in the hepatocyte. The results are proved by the failure to find significant differences in the expression of proteins associated with liver X receptors (SREBP1c, ABCG5 and ABCG8) and the SREBP2 protein (HMG-CoAR and low-density lipoprotein receptor), important transcription factors in the homeostasis and hepatic cholesterol transport. Another effect produced by the overexpression of the NPC1L1 protein in the liver is that it generates an increase in the expression of the hepatic transporter ABCA1 and of lipoproteins rich in apolipoprotein A1, resulting in an increase of plasma cholesterol levels (Temel et al., 2007). This effect, although favoring the treatment and prevention of gallstones, has the adverse reaction of increasing plasma cholesterol. In transgenic mice that overexpress the hepatic NPC1L1 protein, the secretion of biliary cholesterol decreases after treatment with ezetimibe for 4 days (Tang et al., 2011). These findings demonstrated a direct role of hepatic NPC1L1 in regulating biliary cholesterol excretion, but there are only a few basic studies that analyze the protein in conditions of lithiasis.
14 8. Clinical studies of hepatic NPC1L1 protein There are, fortunately, some clinical studies; one of them, for example, carried out in China, included 57 non-obese women with normal levels of plasma lipids and with gallstones, and compared their results with 12 healthy women (Cui et al., 2010). The patients with gallstones showed a reduction in the messenger ribonucleic acid levels (mRNA) of the hepatic NPC1L1 protein; these values were correlated to the mRNA levels of SREBP2, an important regulator of cholesterol transport. Only in women it has been shown that gallstones produce a decrease in the expression of the hepatic NPC1L1 protein; this may be due to the constant cholesterol secretion into the gallbladder. With these results, even though protein expression was evaluated only in females, it is possible to correlate the levels of protein expression to biliary cholesterol secretion; that is, when the expression of hepatic NPC1L1increases, biliary cholesterol secretion decreases, and when the expression of hepatic NPC1L1 decreases, biliary cholesterol secretion increases and there is a greater risk of developing gallstones. Unlike the intestinal protein, the hepatic protein requires the upregulation of its expression to inhibit biliary cholesterol secretion. Two important factors of the pathophysiology of cholesterol gallstones stand out: the intestinal absorption of cholesterol and the secretion of biliary cholesterol (Wang et al., 2009); NPC1L1 proteins are essential for these processes, but the destinations of cholesterol in the body must be controlled, because inhibiting cholesterol at the intestinal or biliary level does not guarantee an optimal removal of it when it is present in high concentrations. The amount of cholesterol in hepatocytes and in plasma lipoproteins ensures that these could be sites of accumulation; in addition, lipoproteins can participate in the development of atherosclerosis. A proper result would be the excretion of cholesterol as
15 neutral sterol in feces. The hepatic NPC1L1 protein requires more research associated with the treatment of cholesterol gallstones.
16 9. NPC1 and NPC2 proteins The NPC1 protein of the hepatocyte plays a major role in the distribution of intracellular cholesterol obtained from lipoproteins by means of endocytosis; this protein plays an important role in the control of biliary cholesterol, the imbalance of which generates cholesterol gallstones (Morales et al., 2010). In mice with a deficiency of the NPC1 protein, cholesterol bile secretion decreases after the administration of a lithogenic diet, which prevents the formation of cholesterol gallstones, compared to control mice; however, these effects produce an accumulation of hepatic cholesterol (Yamanashi et al., 2011). The regulation of the hepatic expression of the NPC1 protein is important in controlling cholesterol bile secretion; thus, its participation as a future therapeutic target is crucial. Currently, there are no clinical studies of this protein associated with cholesterol gallstones. The NPC2 protein is a binding molecule that regulates cholesterol transport in hepatocytes; it is strongly associated with the NPC1L1 protein, which is responsible for down-regulating its expression (Balboa et al., 2012). In mice exhibiting allele alterations of NPC2, the secretion of biliary cholesterol reduces cholesterol bile secretion; this effect prevents the saturation and crystallization of cholesterol in the gallbladder after administering a lithogenic diet. These results are important, but must be researched further. Different studies about Niemann-Pick proteins in the research of cholesterol gallstones were summarized in table 1. Beneficial effects of the intestinal and liver NPC1L1 proteins for the treatment of cholesterol gallstones are showed in figure 1.
17 10. Conclusion NPC1L1 proteins play an important role in the transport of hepatic and intestinal cholesterol; for this reason, it is important to conduct basic and clinical studies aimed to treat and prevent cholesterol gallstones disease, as the drug therapy for it is very limited and there are and numerous risk factors involved.
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22 32. Valasek, M.A., Repa, J.J., Quan, G., Dietschy, J.M., Turley, S.D., 2008. Inhibiting intestinal NPC1L1 activity prevents diet-induced increase in biliary cholesterol in Golden Syrian hamsters. Am. J. Physiol. Gastrointest. Liver. Physiol. 295, G813G822. 33. Van Erpecum, K.J., 2011. Pathogenesis of cholesterol and pigment gallstones: an update. Clin. Res. Hepatol. Gastroenterol. 35, 281-287. 34. Wang, D.Q., Cohen, D.E., Carey, M.C., 2009. Biliary lipids and cholesterol gallstone disease. J. Lipid. Res. 50 Suppl, S406-411. 35. Wang, H.H., Portincasa, P., de Bari, O., Liu, K.J., Garruti, G., Neuschwander-Tetri, B.A., Wang, D.Q., 2013. Prevention of cholesterol gallstones by inhibiting hepatic biosynthesis and intestinal absorption of cholesterol. Eur. J. Clin. Invest. 43, 413426. 36. Wang, H.H., Portincasa, P., Mendez-Sanchez, N., Uribe, M., Wang, D.Q., 2008. Effect of ezetimibe on the prevention and dissolution of cholesterol gallstones. Gastroenterology. 134, 2101-10. 37. Wang, L.J., Song, B.L., 2012. Niemann-Pick C1-Like 1 and cholesterol uptake. Biochim. Biophys. Acta. 1821, 964-972. 38. Wang, L.J., Wang, J., Li, N., Ge, L., Li, B.L., Song, B.L., 2011. Molecular characterization of the NPC1L1 variants identified from cholesterol low absorbers. J. Biol. Chem. 286, 7397-7408. 39. Xie, M., Kotecha, V.R., Andrade, J.D., Fox, J.G., Carey, M.C., 2012. Augmented cholesterol absorption and sarcolemmal sterol enrichment slow small intestinal transit in mice, contributing to cholesterol cholelithogenesis. J. Physiol 590, 18111824.
23 40. Yamanashi, Y., Takada, T., Shoda, J., Suzuki, H., 2011. A novel function of NPC1L1 as a negative regulator of NPC2 protein. Hepatology. 55, 953-964. 41. Yamanashi, Y., Takada, T., Yoshikado, T., Shoda, J., Suzuki, H., 2011. NPC2 regulates biliary cholesterol secretion via stimulation of ABCG5/G8-mediated cholesterol transport. Gastroenterology. 140, 1664-1674. 42. Zúñiga, S., Molina, H., Azocar, L., Amigo, L., Nervi, F., Pimentel, F., Jarufe, N., Arrese, M., Lammert, F., Miquel, J.F., 2008. Ezetimibe prevents cholesterol gallstone formation in mice. Liver. Int. 28: 935-947.
24 Figure captions Fig. 1 Effects of intestinal and hepatic NPC1L1 proteins in the cholesterol gallstones disease.
25 Table 1 Different studies about effect of Niemann-Pick proteins in cholesterol gallstones disease. Type of Description
Model
Reference
Mice
Balboa et al., 2012
- - - -
Cui et al., 2010
- - - -
Jiang et al., 2010
Mice
Morales et al., 2010
Mice
Tang et al., 2011
study Expression of hepatic NPC2 protein reduced In vivo
cholesterol gallstones formation and secretion of biliary cholesterol. A decreased in the expression of hepatic NPC1L1
Clinical
protein
produced
supersaturation
of
biliary
cholesterol in female patients with gallstones. The increased levels of intestinal NPC1L1 protein Clinical
in patients with gallstones produced a high absorption and esterification of cholesterol. The low expression of hepatic NPC1 protein
In vivo inhibited the formation of cholesterol gallstones. Expression of hepatic NPC1L1 protein reduced In vivo
secretion of biliary cholesterol and prevalence of gallstones. The low expression of intestinal NPC1L1 protein
Valasek et al., Hamster
In vivo decreased formation of cholesterol gallstones.
2008/Zúñiga et al., 2008
The low levels of intestinal NPC1L1 protein Clinical/In dissolved cholesterol gallstones in humans. This vivo effect inhibited formation of gallstones in mice.
---/Mice
Wang et al., 2008
26
Intestinal and hepatic Niemann-Pick C1L1 proteins: future therapeutic targets for cholesterol gallstones disease? Ibrahim Guillermo Castro-Torres, Minarda De la O-Arciniega, Gustavo Adolfo Bravo-Duarte, Janeth Gallegos-Estudillo, Miguel Ángel Domínguez-Ortíz, Mariano Martínez-Vázquez www.elsevier.com/locate/ejphar
PII: DOI: Reference:
S0014-2999(14)00103-4 http://dx.doi.org/10.1016/j.ejphar.2014.01.067 EJP69117
To appear in:
European Journal of Pharmacology
Received date: 29 September 2013 Revised date: 13 January 2014 Accepted date: 24 January 2014 Cite this article as: Ibrahim Guillermo Castro-Torres, Minarda De la OArciniega, Gustavo Adolfo Bravo-Duarte, Janeth Gallegos-Estudillo, Miguel Ángel Domínguez-Ortíz, Mariano Martínez-Vázquez, Intestinal and hepatic Niemann-Pick C1L1 proteins: future therapeutic targets for cholesterol gallstones disease?, European Journal of Pharmacology, http://dx.doi.org/10.1016/j. ejphar.2014.01.067 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 Intestinal and hepatic Niemann-Pick C1L1 proteins: future therapeutic targets for cholesterol gallstones disease?
Ibrahim Guillermo Castro-Torresa*, Minarda De la O-Arciniegab, Gustavo Adolfo BravoDuartec, Janeth Gallegos-Estudillod, Miguel Ángel Domínguez-Ortízd, Mariano MartínezVázqueza
a
Instituto de Química. Universidad Nacional Autónoma de México. México, D.F.
b
Área Académica de Farmacia. Instituto de Ciencias de la Salud. Universidad Autónoma
del Estado de Hidalgo. Pachuca de Soto, Hidalgo. México. c
Facultad de Medicina “Lic. Miguel Alemán Valdés”, Universidad Veracruzana, Veracruz,
México. d
Laboratorio de Productos Naturales. Instituto de Ciencias Básicas. Universidad
Veracruzana. Xalapa de Enríquez, Veracruz. México.
Corresponding author: Ibrahim Guillermo Castro-Torres, Prof. Instituto de Química. Universidad Nacional Autónoma de México. Circuito Exterior s/n, Ciudad Universitaria. C.P. 04510. Delegación Coyoacán. México, Distrito Federal. Telephone: (+52) 55 56224403. E-mail: [email protected]
Conflicts of interest: the authors disclose no conflicts.
2 Abstract The formation of cholesterol gallstones is a very complex and polygenic disorder that involves an alteration of the secretion of bile lipids, cholesterol crystallization, important immunological reactions in the gallbladder tissue, formation of biliary sludge composed of mucin, and inadequate gallbladder motility. The search for a therapeutic target is oriented towards decreasing bile secretion and intestinal absorption of cholesterol, in which Niemann-Pick C1L1 (NPC1L1) proteins play an important role. In basic and clinical studies, regulating the expression of these proteins can reduce intestinal, liver, plasma and bile cholesterol levels, a therapeutic effect that would be useful not only for treating the disease, but to prevent it, given the large quantity of risk factors. We discuss these effects in this review and propose NPC1L1 proteins as future therapeutic targets of cholesterol gallstones disease.
Keywords: cholesterol gallstones, NPC1L1 proteins, therapeutic target, gallbladder
3 1. Introduction Cholesterol gallstones disease has many risk factors and is a global problem (Portincasa et al., 2006). Of the different types of gallstones, over 80% are formed by cholesterol; nowadays, this disease is associated with the development of atherosclerosis (MéndezSánchez et al., 2008). There are many cases of gallstones in the United States, Germany, China, Mexico, Chile and Argentina, which lead to the development of numerous gastrointestinal problems and to the performance of thousands of surgeries to remove the gallbladder (Stinton and Shaffer, 2012). Ursodeoxycholic acid is the only drug therapy for cholesterol gallstones, but it can only be used when the stones are made strictly of cholesterol and are in a gallbladder with adequate motility; otherwise, the drug may not exert its mechanism of action (Guarino et al., 2013). Another drug which has been shown to partially dissolve gallstones in humans is ezetimibe, which inhibits the NPC1L1 protein and the intestinal absorption of cholesterol (de Bari et al., 2011; Portincasa et al., 2012). Biliary excretion and intestinal absorption are important processes in the pathophysiology of lithiasis (Portincasa and Wang, 2012), but with respect to treatment, it is important to analyze the mechanisms of action that can disintegrate stones and dissolve cholesterol crystals. The NPC1L1 protein is localized in the proximal jejunum of different species and is also expressed in the liver of humans (Deffieu et al., 2011). In preclinical research, ezetimibe showed a major effect, inhibiting the formation of gallstones in mice; the mechanism of action was found in the intestine, as these rodents do not express the liver protein (Davis and Altmann, 2009). The function of the NPC1L1 protein in the liver has not been fully described yet; however, it takes part in regulating the concentration and transport of cholesterol in the intestine (Pramfalk et al., 2011, Wang and Song, 2012, Castro-Torres
4 and Bravo-Duarte, 2013), making it an essential element in the pathophysiology of biliary lithiasis.
5 2. Pathophysiology of gallstones The search for pharmacological therapies is complicated due to the complexity of the steps in which a gallstone is formed. These steps involve imbalances in the secretion of biliary cholesterol, the crystallization/nucleation of cholesterol, inflammatory reactions of the gallbladder, and the alteration of the secretion of mucin proteins; some authors also argue the importance of intestinal and gallbladder motility (Maurer et al., 2009; Castro-Torres, 2012; Krawczyk et al., 2012). When the secretion of bile lipids becomes altered, cholesterol can saturate the gallbladder and produce imbalances. In this first phase, the most important proteins are family members ATP-binding cassette transporters (ABC): ABCB11 (bile salts), ABCB4 (phospholipids), ABCG5 and ABCG8 (cholesterol transporters) (CastroTorres and Martínez-Vázquez, 2013). Regarding the transport of cholesterol, several nuclear receptors and metabolism enzymes are involved at the hepatocyte level: farnesoid receptor, liver X receptors, pregnane receptor, sterol response element binding protein SREBP-1 and SREBP-2, NPC1L1 protein, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoAR) and cholesterol 7α-hydroxylase. In addition to hepatocytes, the transport of biliary cholesterol before the lipid reaches ABCG5/8 heterodimers must be considered; in this, the high-density lipoprotein and its scavenger receptor-1 are very important (Chuang et al., 2013; Di Ciaula et al 2013). Each of these molecules deserves to be researched to look for preventive and therapeutic breakthroughs. When the vesicle has high cholesterol concentrations, the lipid may undergo crystallization, an important and limiting physiochemical change in the development of the gallstone (Abeysuriya et al 2010). Mucin proteins (5B and 5AC primarily) and other pronuclear factors are involved at this pathophysiological stage (Kim et al., 2012); here, too, there could be a therapeutic target to prevent the change of the physical state of cholesterol. The motility of the gallbladder can
6 also be studied to look for a treatment; under conditions of lithiasis, the emptying and filling of the gallbladder becomes altered, favoring the growth of stones. The hormone cholecystokinin and its main type 1 receptor, control this movement of the gallbladder (Xie et al., 2012). It has been shown that in mice susceptible to the development of lithiasis, a deficiency of the cholecystokinin receptor, promotes the development of gallstones, even after treatment with salts of ursodeoxycholic acid, the most important bile acid for dissolving cholesterol gallstones (Nihei et al., 2011). These data demonstrate the importance of the receptor in the pathophysiology of lithiasis and suggest it as a potential therapeutic target. One of the most interesting sites for gallstones therapy is the small intestine, where the absorption of cholesterol takes place. Proteins NPC1L1, ABCG5 and ABCG8 are expressed in the brush border of enterocytes, which are involved in the transport of cholesterol (Davis et al., 2011); the lipid comes mostly from the diet, so a high fat intake increases the risk of developing hypercholesterolemia and gallbladder lithiasis. Inhibiting NPC1L1 expression inhibits the intestinal absorption of cholesterol, preventing the development of gallstones and reducing plasma cholesterol levels (Tang et al., 2011); the drug ezetimibe has the ability to inhibit the expression of protein NPC1L1 and this route is of particular interest for the search for a gallstones treatment. With respect to intestinal absorption, the enzymes that allow the transport of cholesterol are very important: cholesteryl hydrolase and acyl-coenzyme A: cholesterol acyltransferase (ACAT2).The first enzyme hydrolyzes cholesterol so that it can pass through the enterocyte, and the second one esterifies it so that it can be transported in chylomicrons to the liver (Grenier et al., 2012).
7 3. Intestinal NPC1L1 protein and cholesterol transport This protein is expressed in the cells that form the brush border of the enterocytes; the region where its expression is higher is the proximal jejunum, a site of cholesterol absorption (Davis and Altmann, 2009). The human protein contains 1332 amino acids and 13 transmembrane domains, of which 5 function as sterol detection sites (Wang and Song, 2012). Dietary cholesterol is absorbed in the intestine and reaches the liver via systemic circulation; thus, the participation of the NPC1L1 protein is very important because it can determine the amount of cholesterol circulating to the liver and allow for it to be disposed as neutral sterol. Biliary cholesterol secretion may depend on intestinal absorption and hepatic cholesterol concentration. The intestine receives cholesterol mainly from food, bile secretion and, in a lesser scale, from the desquamation suffered by its epithelium (Davis et al., 2011). The transport of cholesterol through the NPC1L1 protein is highly selective, unlike what happens with other sterols; it occurs via vesicular endocytosis mediated by the protein clathrin (Wang et al., 2011). Another molecule required for intestinal cholesterol absorption, called caveolin-1, has been identified (Tang et al., 2011). Cholesterol reaches the brush border membrane of enterocyte in mixed micelles or phospholipid vesicles (liposomes); these transport complexes must release it so it can be binded by specific domains of the protein NPC1L1; if the lipid is esterified, it must be hydrolyzed by the cholesterol ester hydrolase enzyme so that it can be absorbed (Grenier et al., 2012). After cholesterol passes through the enterocyte membrane, it heads to the endoplasmic reticulum to be esterified by the ACAT2; subsequent to this, it is transported in the chylomicrons circulating in the lymphatic system of the intestine and the thoracic duct that end their route in the systemic circulation, where they reach the liver as chylomicron remnants (Castro-Torres and Martínez-Vázquez, 2013).
8 4. Basic research of intestinal NPC1L1 protein Important basic works have studied the expression of NPC1L1 protein with respect to the prevention of gallstones. Few studies focus on gallstones treatment because they tend to take into account cost-benefit considerations during the development of the disease. C57BL/6 and C57L/J mice are important models for the study of cholesterol gallstones and these strains have been used in important studies about the expression of NPC1L1 proteins in the presence of gallstones. Ezetimibe is a drug that blocks the expression of NPC1L1 and this causes a decrease in cholesterol absorption by the small intestine (Wang et al., 2013). This drug decreases the amount of cholesterol delivered to the liver and bile, thereby reducing the amount of cholesterol available for the formation of gallstones (Stein et al., 2012). An important preclinical study used male C57BL/6 mice in which gallstones were induced by a diet enriched in cholesterol and cholic acid. Mice were administered with ezetimibe at a dose of 5 mg/kg for 14 days. Ezetimibe administration reduced intestinal cholesterol absorption by 90% in control animals and by 35% in mice with cholesterol gallstones; the treatment also prevented the formation of cholesterol crystals and gallstones. Mice treated with the drug exhibited a 60% reduction in biliary cholesterol saturation index. There was a significant decrease in the expression of the NPC1L1 protein in mice fed with lithogenic diet due to the effect of ezetimibe (Zúñiga et al., 2008). The authors demonstrated that the decrease of intestinal NPC1L1 expression could be a molecular pathway for the prevention of gallstones. In another preclinical study, C57L/J mice were used to study the prevention of lithiasis. C57L/J mice fed a lithogenic diet (1% cholesterol and 0.1% cholic acid) developed gallstones in less than a month; in this model, the administration of ezetimibe at doses of 8
9 mg/kg/day reduced the expression of intestinal NPC1L1 protein. This effect produced a decrease in the intestinal absorption of cholesterol (5.0±1.4%), compared with mice afflicted with gallstones (62.5 ± 4.6%); in addition, it decreased biliary cholesterol secretion and improved gallbladder motility with no alteration of bile salt content (80.70% molar) (Wang et al., 2008). Considering that an important process of the pathophysiology of lithiasis takes place in the gallbladder, the effects caused by the inhibition of NPC1L1 may be employed in the treatment and prevention of the disease. If we speak of lithiasis prevention, decreasing intestinal cholesterol by inhibiting NPC1L1 prevents the circulation of high concentrations of the lipid towards the liver; this would also reduce cholesterol secretion in bile, preventing its accumulation in the gallbladder. With regard to treatment, when gallstones have already formed, its main component is crystallized cholesterol, but the low expression of intestinal NPC1L1 reduces bile secretion, improves motility and does not alter the content of bile salts. The latter solutes are the most abundant in the bile, and when they are in a gallbladder with adequate motility, they have the ability to emulsify the cholesterol of the gallstones, causing their partial or complete disintegration. This mechanism of action exerted by bile salts is the mechanism of ursodeoxycholic acid, but in this case, such effects are produced by the NPC1L1 protein of the small intestine, which, from a distant site to the gallbladder, controls the transport of cholesterol, preventing supersaturation in bile. A study in Golden Syrian hamsters, which were fed a high fat diet and ezetimibe at doses of 2 mg/kg/day, demonstrated that biliary cholesterol secretion decreased as a result of the pharmacological effect, which was the decrease in intestinal NPC1L1 expression. At this dose of ezetimiba, cholesterol absorption fell by 82%, fecal neutral sterol excretion increased 5.3-fold, and hepatic and intestinal cholesterol synthesis increased more than
10 twofold, but there were no significant changes in either fecal bile acid excretion or biliary lipid composition (Valasek et al., 2008). It is necessary to analyze more proteins involved in cholesterol transport and metabolism in order to find different mechanisms of action. The fact that a drug with the property of inhibiting the expression of the intestinal NPC1L1 protein already exists does not eliminate the need to research the effects that this protein can have on gallstones; it is also important to consider the liver protein, which, because of its place of expression, prevents ezetimibe from acting in the same way, and if it could do so, its effects on the concentration of cholesterol would not be the same, since the intestine and the liver have their own cell medium and their own nuclear receptors involved in the physiological regulation of the lipid.
11 5. Clinical studies of intestinal NPC1L1 protein A clinical study in 12 Chinese patients with cholesterol gallstones and 31 control patients demonstrated that, as a consequence of the disease, the mRNA levels of proteins NPC1L1 and ACAT2 (the primary enzyme responsible for esterified cholesterol) increased, resulting in an overexpression of them, whereas in the transport ABCG5/8 proteins no changes were observed. These results were obtained from protein analysis in jejunal biopsies (Jiang et al., 2009). The data indicate that biliary lithiasis produces a high concentration of intestinal cholesterol and, as the lipid circulates mostly to the liver, it must first be transported (NPC1L1 overexpression) and then esterified to enter into chylomicrons (ACAT2 overexpression); thus the downregulation of the expression of intestinal NPC1L1 could counter these cholesterol imbalances. Currently, the drug ezetimibe has the function of decreasing the intestinal absorption of cholesterol, but there is only one clinical study associated to the treatment of cholesterol gallstones, where the action of the drug (20 mg/kg/day) reduces biliary cholesterol secretion in sick patients, decreasing the size of the gallstones (Wang et al., 2008).
12 6. Hepatic NPC1L1 protein and cholesterol transport This protein is expressed in the canalicular membrane of the hepatocyte, and its role in cholesterol transport has not yet been fully explained. Within its participation in homeostasis, it regulates the expression of the Niemann-Pick C2 (NPC2) protein and involves the participation of many transcription factors (Yamanashi et al., 2011). The cholesterol linked to this protein is the one coming from the systemic circulation and that which is synthesized de novo in the liver (Temel et al., 2007). Different studies have determined that cholesterol transport throughout the hepatic NPC1L1 protein is very complex, carried out it by vesicular endocytosis mediated by a clathrin AP2 complex. The hepatic NPC1L1 protein has specific microdomains where the lipid-protein interaction can be carried out. This type of transport is carried out by lipid rafts and involves flotillin proteins 1 and 2 (Cianciola et al., 2011). The cell medium of the hepatic NPC1L1 protein and the type of genetic regulation it is subjected to, determine that its effect on cholesterol in the hepatocyte is not the same as that on intestinal cholesterol; therefore, its participation as a therapeutic target in the treatment of cholesterol gallstones must be carefully differentiated.
13 7. Basic research of hepatic NPC1L1 protein Rodents do not express the protein NPC1L1 in the liver, but it has been possible to generate transgenic models that can do it. It was determined in rat livers capable of expressing the human NPC1L1 protein, that with the increase in the expression of the liver protein, the secretion of biliary cholesterol decreases and hepatic reabsorption occurs, but it has also been shown that there is a minimal increase of cholesterol in the liver (30), indicating that cholesterol does not necessarily accumulate in the hepatocyte. The results are proved by the failure to find significant differences in the expression of proteins associated with liver X receptors (SREBP1c, ABCG5 and ABCG8) and the SREBP2 protein (HMG-CoAR and low-density lipoprotein receptor), important transcription factors in the homeostasis and hepatic cholesterol transport. Another effect produced by the overexpression of the NPC1L1 protein in the liver is that it generates an increase in the expression of the hepatic transporter ABCA1 and of lipoproteins rich in apolipoprotein A1, resulting in an increase of plasma cholesterol levels (Temel et al., 2007). This effect, although favoring the treatment and prevention of gallstones, has the adverse reaction of increasing plasma cholesterol. In transgenic mice that overexpress the hepatic NPC1L1 protein, the secretion of biliary cholesterol decreases after treatment with ezetimibe for 4 days (Tang et al., 2011). These findings demonstrated a direct role of hepatic NPC1L1 in regulating biliary cholesterol excretion, but there are only a few basic studies that analyze the protein in conditions of lithiasis.
14 8. Clinical studies of hepatic NPC1L1 protein There are, fortunately, some clinical studies; one of them, for example, carried out in China, included 57 non-obese women with normal levels of plasma lipids and with gallstones, and compared their results with 12 healthy women (Cui et al., 2010). The patients with gallstones showed a reduction in the messenger ribonucleic acid levels (mRNA) of the hepatic NPC1L1 protein; these values were correlated to the mRNA levels of SREBP2, an important regulator of cholesterol transport. Only in women it has been shown that gallstones produce a decrease in the expression of the hepatic NPC1L1 protein; this may be due to the constant cholesterol secretion into the gallbladder. With these results, even though protein expression was evaluated only in females, it is possible to correlate the levels of protein expression to biliary cholesterol secretion; that is, when the expression of hepatic NPC1L1increases, biliary cholesterol secretion decreases, and when the expression of hepatic NPC1L1 decreases, biliary cholesterol secretion increases and there is a greater risk of developing gallstones. Unlike the intestinal protein, the hepatic protein requires the upregulation of its expression to inhibit biliary cholesterol secretion. Two important factors of the pathophysiology of cholesterol gallstones stand out: the intestinal absorption of cholesterol and the secretion of biliary cholesterol (Wang et al., 2009); NPC1L1 proteins are essential for these processes, but the destinations of cholesterol in the body must be controlled, because inhibiting cholesterol at the intestinal or biliary level does not guarantee an optimal removal of it when it is present in high concentrations. The amount of cholesterol in hepatocytes and in plasma lipoproteins ensures that these could be sites of accumulation; in addition, lipoproteins can participate in the development of atherosclerosis. A proper result would be the excretion of cholesterol as
15 neutral sterol in feces. The hepatic NPC1L1 protein requires more research associated with the treatment of cholesterol gallstones.
16 9. NPC1 and NPC2 proteins The NPC1 protein of the hepatocyte plays a major role in the distribution of intracellular cholesterol obtained from lipoproteins by means of endocytosis; this protein plays an important role in the control of biliary cholesterol, the imbalance of which generates cholesterol gallstones (Morales et al., 2010). In mice with a deficiency of the NPC1 protein, cholesterol bile secretion decreases after the administration of a lithogenic diet, which prevents the formation of cholesterol gallstones, compared to control mice; however, these effects produce an accumulation of hepatic cholesterol (Yamanashi et al., 2011). The regulation of the hepatic expression of the NPC1 protein is important in controlling cholesterol bile secretion; thus, its participation as a future therapeutic target is crucial. Currently, there are no clinical studies of this protein associated with cholesterol gallstones. The NPC2 protein is a binding molecule that regulates cholesterol transport in hepatocytes; it is strongly associated with the NPC1L1 protein, which is responsible for down-regulating its expression (Balboa et al., 2012). In mice exhibiting allele alterations of NPC2, the secretion of biliary cholesterol reduces cholesterol bile secretion; this effect prevents the saturation and crystallization of cholesterol in the gallbladder after administering a lithogenic diet. These results are important, but must be researched further. Different studies about Niemann-Pick proteins in the research of cholesterol gallstones were summarized in table 1. Beneficial effects of the intestinal and liver NPC1L1 proteins for the treatment of cholesterol gallstones are showed in figure 1.
17 10. Conclusion NPC1L1 proteins play an important role in the transport of hepatic and intestinal cholesterol; for this reason, it is important to conduct basic and clinical studies aimed to treat and prevent cholesterol gallstones disease, as the drug therapy for it is very limited and there are and numerous risk factors involved.
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23 40. Yamanashi, Y., Takada, T., Shoda, J., Suzuki, H., 2011. A novel function of NPC1L1 as a negative regulator of NPC2 protein. Hepatology. 55, 953-964. 41. Yamanashi, Y., Takada, T., Yoshikado, T., Shoda, J., Suzuki, H., 2011. NPC2 regulates biliary cholesterol secretion via stimulation of ABCG5/G8-mediated cholesterol transport. Gastroenterology. 140, 1664-1674. 42. Zúñiga, S., Molina, H., Azocar, L., Amigo, L., Nervi, F., Pimentel, F., Jarufe, N., Arrese, M., Lammert, F., Miquel, J.F., 2008. Ezetimibe prevents cholesterol gallstone formation in mice. Liver. Int. 28: 935-947.
24 Figure captions Fig. 1 Effects of intestinal and hepatic NPC1L1 proteins in the cholesterol gallstones disease.
25 Table 1 Different studies about effect of Niemann-Pick proteins in cholesterol gallstones disease. Type of Description
Model
Reference
Mice
Balboa et al., 2012
- - - -
Cui et al., 2010
- - - -
Jiang et al., 2010
Mice
Morales et al., 2010
Mice
Tang et al., 2011
study Expression of hepatic NPC2 protein reduced In vivo
cholesterol gallstones formation and secretion of biliary cholesterol. A decreased in the expression of hepatic NPC1L1
Clinical
protein
produced
supersaturation
of
biliary
cholesterol in female patients with gallstones. The increased levels of intestinal NPC1L1 protein Clinical
in patients with gallstones produced a high absorption and esterification of cholesterol. The low expression of hepatic NPC1 protein
In vivo inhibited the formation of cholesterol gallstones. Expression of hepatic NPC1L1 protein reduced In vivo
secretion of biliary cholesterol and prevalence of gallstones. The low expression of intestinal NPC1L1 protein
Valasek et al., Hamster
In vivo decreased formation of cholesterol gallstones.
2008/Zúñiga et al., 2008
The low levels of intestinal NPC1L1 protein Clinical/In dissolved cholesterol gallstones in humans. This vivo effect inhibited formation of gallstones in mice.
---/Mice
Wang et al., 2008
26